U.S. patent number RE39,580 [Application Number 10/061,082] was granted by the patent office on 2007-04-17 for insect-resistant transgenic plants.
This patent grant is currently assigned to Monsanto Technology LLC. Invention is credited to Susan M. Brussock, James W. Bryson, Leigh H. English, Caroline A. Kulesza, Thomas M. Malvar, Charles Romano, Stephen L. Slatin, Michael A. Von Tersch, Frederick S. Walters.
United States Patent |
RE39,580 |
English , et al. |
April 17, 2007 |
Insect-resistant transgenic plants
Abstract
The invention provides transgenic plants and transformed host
cells which express modified cry 3B genes with enhanced toxicity to
Coleopteran insects. Also disclosed are methods of making and using
these transgenic plants, methods of making recombinant host cells
expressing these .delta.-endotoxins, and methods of killing insects
such as Colorado potato beetle (Leptinotarsa decemlineata),
southern corn rootworm (Diabrotica undecimpunctata howardi Barber)
and western corn rootworm (Diabrotica virgifera virgifera
LeConte.
Inventors: |
English; Leigh H.
(Chesterfield, MO), Brussock; Susan M. (New Hope, PA),
Malvar; Thomas M. (Troy, MO), Bryson; James W.
(Langhorne, PA), Kulesza; Caroline A. (Cranbury, NJ),
Walters; Frederick S. (Raleigh, NC), Slatin; Stephen L.
(New York, NY), Von Tersch; Michael A. (Trenton, NJ),
Romano; Charles (Chesterfield, MO) |
Assignee: |
Monsanto Technology LLC (St.
Louis, MO)
|
Family
ID: |
25542921 |
Appl.
No.: |
10/061,082 |
Filed: |
January 31, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
08996441 |
Dec 18, 1997 |
06023013 |
Feb 8, 2000 |
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Current U.S.
Class: |
800/302;
435/252.3; 435/418; 800/279 |
Current CPC
Class: |
C07K
14/325 (20130101) |
Current International
Class: |
A01H
5/00 (20060101); A01H 5/10 (20060101); C12N
1/21 (20060101); C12N 15/82 (20060101) |
Field of
Search: |
;800/302,279,292,293,294
;435/418,252.3,252.31,469,470 ;536/23.71 |
References Cited
[Referenced By]
U.S. Patent Documents
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|
Primary Examiner: Kubelik; Anne
Attorney, Agent or Firm: Ball; Timothy K. Howrey LLP
Claims
What is claimed is:
1. A transgenic plant having incorporated into its genome a
.Iadd.modified cry3B* .Iaddend.gene that encodes an amino acid
sequence selected from the group consisting of SEQ ID NO:2, SEQ ID
NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ
ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,
SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID
NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ
ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60,
SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID
NO:70, SEQ ID NO:100, and SEQ ID NO:108.
2. A transgenic plant having incorporated into its genome a
.Iadd.modified cry3B* .Iaddend.gene comprising a nucleic acid
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID
NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ
ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31,
SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID
NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ
ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59,
SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID
NO:69, SEQ ID NO:99, and SEQ ID NO:107.
3. A progeny or seed from the transgenic plant of claim 1 or claim
2 comprising .[.a.]. .Iadd.the .Iaddend.modified cry3B* gene.
4. A seed from the progeny of claim 3 comprising .[.a.]. .Iadd.the
.Iaddend.modified cry3B* gene.
5. A plant from the .Iadd.progeny or .Iaddend.seed of claim 3 .[.or
claim 4.]. comprising .[.a.]. .Iadd.the .Iaddend.modified cry3B*
gene.
6. A host cell having an NRRL accession number selected from the
group consisting of NRRL B-21744, NRRL B-21745, NRRL B-21746, NRRL
B-21747, NRRL B-21748, NRRL B-21749, NRRL B-21750, NRRL B-21751,
NRRL B-21752, NRRL B-21753, NRRL B-21754, NRRL B-21755, NRRL
B-21756, NRRL B-21757, NRRL B-21758, NRRL B-21759, NRRL B-21760,
NRRL B-21761, NRRL B-21762, NRRL B-21763, NRRL B-21764, NRRL
B-21765, NRRL B-21766, NRRL B-21767, NRRL B-21768, NRRL B-21769,
NRRL B-21770, NRRL B-21771, NRRL B-21772, NRRL B-21773, NRRL
B-21774, NRRL B-21775, NRRL B-21776, NRRL B-21777, NRRL B-21778,
and NRRL B-21779.
7. A host cell selected from the group consisting of an EG11221,
EG11222, EG11223, EG11224, EG11225, EG11226, EG11227, EG11228,
EG11229, EG11230, EG11231, EG11232, EG11233, EG11234, EG11235,
EG11236, EG11237, EG11238, EG11239, EG11241, EG11242, EG11032,
EG11035, EG11036, EG11046, EG11048, EG11051, EG11057, EG11058,
EG11081, EG11082, EG11083, EG11084, EG11095, and an EG11098
cell.
8. A transformed bacterial or plant host cell comprising a modified
cry3B* gene that encodes a modified Cry3B* protein having enhanced
insecticidal activity against a Coleopteran insect pest relative to
an unmodified .[.Cry3B*.]. .Iadd.Cry3B .Iaddend.protein.Iadd.,
wherein said modified Cry3B* protein comprises the amino acid
sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ
ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,
SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID
NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ
ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46,
SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID
NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ
ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, or SEQ ID
NO:108.Iaddend..
9. The host cell of claim 8, wherein said host cell is an E. coli,
B. thuringiensis, A. tumefaciens, B. subtilis, B. megaterium, B.
cereus, Salmonella spp., or Pseudomonas spp. cell.
10. The host cell of claim 9, wherein said B. thuringiensis cell
has an NRRL accession number selected from the group consisting of
NRRL B-21744, NRRL B-21745, NRRL B-21746, NRRL B-21747, NRRL
B-21748, NRRL B-21749, NRRL B-21750, NRRL B-21751, NRRL B-21752,
NRRL B-21753, NRRL B-21754, NRRL B-21755, NRRL B-21756, NRRL
B-21757, NRRL B-21758, NRRL B-21759, NRRL B-21760, NRRL B-21761,
NRRL B-21762, NRRL B-21763, NRRL B-21764, NRRL B-21765, NRRL
B-21766, NRRL B-21767, NRRL B-21768, NRRL B-21769, NRRL B-21770,
NRRL B-21771, NRRL B-21772, NRRL B-21773, NRRL B-21774, NRRL
B-21775, NRRL B-21776, NRRL B-21777, NRRL B-21778, and NRRL
B-21779.
11. The host cell of claim 8, wherein said host cell is a plant
cell selected from the group consisting of a grain, tree, legume,
vegetable, fruit, berry, nut, citrus, grass, cactus, succulent, or
ornamental plant cell.
12. The host cell of claim 11, wherein said plant cell is a corn,
rice, tobacco, alfalfa, soybean, sorghum, potato, tomato, flax,
canola, sunflower, cotton, flax, kapok, wheat, oat, barley, or rye
cell.
13. The host cell of claim 12, wherein said plant cell is a corn,
rice, tobacco, alfalfa, soybean, canola, sunflower, cotton, or
wheat cell.
14. The host cell of claim 8, wherein said gene is introduced into
said cell by a recombinant vector, virus, cosmid, phagemid, phage,
or plasmid.
15. The host cell of claim 8, wherein said gene is introduced into
said cell by electroporation, transformation, conjugation,
microprojectile bombardment, direct DNA injection, or
transfection.
.[.16. The host cell of claim 8, wherein said modified Cry3B*
protein comprises the amino acid sequence of SEQ ID NO:2, SEQ ID
NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ
ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,
SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID
NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ
ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60,
SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID
NO:70, SEQ ID NO:100, or SEQ ID NO:108..].
17. The host cell of claim .[.16.]. .Iadd.8.Iaddend., wherein said
.Iadd.modified Cry3B* .Iaddend.protein is encoded by the nucleic
acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ
ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35,
SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID
NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ
ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63,
SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99, or SEQ ID
NO:107.
18. The host cell of claim 8, wherein said cell is comprised within
a transgenic plant.
19. The host cell of claim 18, wherein said cell produces a
polypeptide having insecticidal activity against Colorado potato
beetle, corn borer or corn rootworm.
20. The host cell of claim 19, wherein said cell produces a
polypeptide having insecticidal activity against Leptinotarsa
decemlineata, Diabrotica undecimpunctata howardi, Diabrotica
virgifera, or Diabrotica virgifera virgifera.
21. A transformed plant cell comprising a modified cry3B* gene that
encodes a modified Cry3B* polypeptide having an insecticidal
activity which is enhanced against a target insect, compared to a
plant cell comprising a native or unmodified cry3B gene.Iadd.,
wherein said modified Cry3B* polypeptide comprises the amino acid
sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ
ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,
SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID
NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ
ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46,
SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID
NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ
ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, or SEQ ID
NO:108.Iaddend..
22. The transformed plant cell of claim 21, wherein said modified
cry3B* gene produces a modified Cry3B* polypeptide which has
enhanced insecticidal activity against Colorado potato beetle, corn
borer or corn rootworm.
23. A method of preparing a transgenic plant resistant to
infestation by a Coleopteran insect, .[.comprising.]. .Iadd.wherein
the method comprises .Iaddend.transforming said plant with a
nucleic acid segment that encodes an amino acid sequence selected
from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ
ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34,
SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID
NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ
ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,
SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID
NO:100, and SEQ ID NO:108.
24. A method of killing a Coleopteran insect, .[.comprising.].
.Iadd.wherein the method comprises .Iaddend.providing to said
insect one or more plant cells transformed with a nucleic acid
segment that encodes an amino acid sequence selected from the group
consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,
SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID
NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ
ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36,
SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID
NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ
ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64,
SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, and SEQ ID
NO:108, wherein said insect is killed by ingesting one or more of
said transformed plant cells.
25. A method of preparing a plant seed resistant to Coleopteran
insect attack, said method comprising the steps of: (a)
transforming a plant cell with a nucleic acid segment comprising a
modified cry3B* gene that encodes an amino acid sequence selected
from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ
ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34,
SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID
NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ
ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,
SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID
NO:100, and SEQ ID NO:108 to produce a transformed plant cell; (b)
producing a transgenic plant from said transformed plant cell; and
(c) obtaining from said transgenic plant, a seed resistant to
attack by said Coleopteran insect.
26. The method of claim 25 wherein step (a) comprises transforming
said plant cell by electroporation, protoplast generation, direct
transfer of DNA into pollen or an embryo, Agrobacterium-mediated
transformation, particle bombardment, or microprojectile
bombardment.
27. The method of claim 25, wherein step (b) comprises generation
of plant cells from said transformed plant cell, and regeneration
of said transgenic plant from said plant cells.
Description
1.0 BACKGROUND OF THE INVENTION
1.1 Field of the Invention
This invention relates to transgenic plants which express
bacterially-derived proteins which are toxic to Coleopteran insects
such as Colorado potato beetle (Leptinotarsa decemlineata),
southern corn rootworm (Diabrotica undecimpunctata howardi Barber)
and western corn rootworm (Diabrotica virgifera virgifera
LeConte).
1.2 Description of the Related Art
Almost all field crops, plants, and commercial farming areas are
susceptible to attack by one or more insect pests. Particularly
problematic are Coleopteran and Lepidoptern pests. For example,
vegetable and cole crops such as artichokes, kohlrabi, arugula,
leeks, asparagus, lentils, beans, lettuce (e.g., head, leaf,
romaine), beets, bok choy, malanga, broccoli, melons (e.g.,
muskmelon, watermelon, crenshaw, honeydew, cantaloupe), brussels
sprouts, cabbage, cardoni, carrots, napa, cauliflower, okra,
onions, celery, parsley, chick peas, parsnips, chicory, peas,
chinese cabbage, peppers, collards, potatoes, cucumber, pumpkins,
cucurbits, radishes, dry bulb onions, rutabaga, eggplant, salsify,
escarole, shallots, endive, soybean, garlic, spinach, green onions,
squash, greens, sugar beets, sweet potatoes, turnip, swiss chard,
horseradish, tomatoes, kale, turnips, and a variety of spices are
sensitive to infestation by one or more of the following insect
pests: alfalfa looper, armyworm, beet armyworm, artichoke plume
moth, cabbage budworm, cabbage looper, cabbage webworm, corn
earworm, celery leafeater, cross-striped cabbageworm, european corn
borer, diamondback moth, green cloverworm, imported cabbageworm,
melonworm, omnivorous leafroller, pickleworm, rindworm complex,
saltmarsh caterpillar, soybean looper, tobacco budworm, tomato
fruitworm, tomato horuworm, tomato pinworm, velvetbean caterpillar,
and yellowstriped armyworm. Likewise, pasture and hay crops such as
alfalfa, pasture grasses and silage are often attacked by such
pests as armyworm, beef armyworm, alfalfa caterpillar, European
skipper, a variety of loopers and webworms, as well as
yellowstriped armyworms.
Fruit and vine crops such as apples, apricots, cherries,
nectarines, peaches, pears, plums, prunes, quince almonds,
chestnuts, filberts, pecans, pistachios, walnuts, citrus,
blackberries, blueberries, boysenberries, cranberries, currants,
loganberries, raspberries, strawberries, grapes, avocados, bananas,
kiwi, persimmons, pomegranate, pineapple, tropical fruits are often
susceptible to attack and defoliation by achema sphinx moth,
amorbia, armyworm, citrus cutworm, banana skipper, blackheaded
fireworm, blueberry leafroller, cankerworm, cherry fruitworm,
citrus cutworm, cranberry girdler, eastern tent caterpillar, fall
webworm, fall webworm, filbert leafroller, filbert webworm, fruit
tree leafroller, grape berry moth, grape leaffolder, grapeleaf
skeletonizer, green fruitworm, gumrnososbatrachedra commosae, gypsy
moth, hickory shuckworm, hormworms, loopers, navel orangeworm,
obliquebanded leafroller, omnivorous leafroller, omnivorous looper,
orange tortrix, orangedog, oriental fruit moth, pandemis
leafroller, peach twig borer, pecan nut casebearer, redbanded
leafroller, redhumped caterpillar, roughskinned cutworm, saltmarsh
caterpillar, spanworm, tent caterpillar, thecla-thecla basillides,
tobacco budworm, tortrix moth, tufted apple budmoth, variegated
leafroller, walnut caterpillar, western tent caterpillar, and
yellowstriped armyworm.
Field crops such as canola/rape seed, evening primrose, meadow
foam, corn (field, sweet, popcorn), cotton, hops, jojoba, peanuts,
rice, safflower, small grains (barley, oats, rye, wheat, etc.),
sorghum, soybeans, sunflowers, and tobacco are often targets for
infestation by insects including armyworm, asian and other corn
borers, banded sunflower moth, beet armyworm, bollworm, cabbage
looper, corn rootworm (including southern and western varieties),
cotton leaf perforator, diamondback moth, european corn borer,
green cloverworm, headmoth, headworm, imported cabbageworm, loopers
(including Anacamptodes spp.), obliquebanded leafroller, omnivorous
leaftier, podworm, podworm, saltmarsh caterpillar, southwestern
corn borer, soybean looper, spotted cutworm, sunflower moth,
tobacco budworm, tobacco hornworm, velvetbean caterpillar,
Bedding plants, flowers, ornamentals, vegetables and container
stock are frequently fed upon by a host of insect pests such as
armyworm, azalea moth, beet armyworm, diamondback moth, ello moth
(hornworm), Florida fern caterpillar, Io moth, loopers, oleander
moth, omnivorous leafroller, omnivorous looper, and tobacco
budworm.
Forests, fruit, ornamental, and nut-bearing trees, as well as
shrubs and other nursery stock are often susceptible to attack from
diverse insects such as bagworm, blackheaded budworm, browntail
moth, california oakworm, douglas fir tussock moth, elm spanworm,
fall webworm, fruittree leafroller, greenstriped mapleworm, gypsy
moth, jack pine budworm, mimosa webworm, pine butterfly, redhumped
caterpillar, saddleback caterpillar, saddle prominent caterpillar,
spring and fall cankerworm, spruce budworm, tent caterpillar,
tortrix, and western tussock moth. Likewise, turf grasses are often
attacked by pests such as armyworm, sod webworm, and tropical sod
webworm.
Because crops of commercial interest are often the target of insect
attack, environmentally-sensitive methods for controlling or
eradicating insect infestation are desirable in many instances.
This is particularly true for farmers, nurserymen, growers, and
commercial and residential areas which seek to control insect
populations using eco-friendly compositions.
The most widely used environmentally-sensitive insecticidal
formulations developed in recent years have been composed of
microbial pesticides derived from the bacterium Bacillus
thuringiensis. B. thuringiensis is a Gram-positive bacterium that
produces crystal proteins or inclusion bodies which are
specifically toxic to certain orders and species of insects. Many
different strains of B. thuringiensis have been shown to produce
insecticidal crystal proteins. Compositions including B.
thuringiensis strains which produce insecticidal proteins have been
commercially-available and used as environmentally-acceptable
insecticides because they are quite toxic to the specific target
insect, but are harmless to plants and other non-targeted
organisms.
1.2.1 .delta.-Endotoxins
.delta.-endotoxins are used to control a wide range of leaf-eating
caterpillars and beetles, as well as mosquitoes. These
proteinaceous parasporal crystals, also referred to as insecticidal
crystal proteins, crystal proteins, Bt inclusions, crystaline
inclusions, inclusion bodies, and Bt toxins, are a large collection
of insecticidal proteins produced by B. thuringiensis that are
toxic upon ingestion by a susceptible insect host. Over the past
decade research on the structure and function of B. thuringiensis
toxins has covered all of the major toxin categories, and while
these toxins differ in specific structure and function, general
similarities in the structure and function are assumed. Based on
the accumulated knowledge of B. thuringiensis toxins, a generalized
mode of action for B. thuringiensis toxins has been created and
includes: ingestion by the insect, solubilization in the insect
midgut (a combination stomach and small intestine), resistance to
digestive enzymes sometimes with partial digestion actually
"activating" the toxin, binding to the midgut cells, formation of a
pore in the insect cells and the disruption of cellular homeostasis
(English and Slatin, 1992).
1.2.2 Genes Encoding Crystal Proteins
Many of the .delta.-endotoxins are related to various degrees by
similarities in their amino acid sequences. Historically, the
proteins and the genes which encode them were classified based
largely upon their spectrum of insecticidal activity. The review by
Hofte and Whiteley (1989) discusses the genes and proteins that
were identified in B. thuringiensis prior to 1990, and sets forth
the nomenclature and classification scheme which has traditionally
been applied to B. thuringiensis genes and proteins. cryI genes
encode lepidopteran-toxic CryI proteins. cryII genes encode CryII
proteins that are toxic to both lepidopterans and dipterans. cryIII
genes encode coleopteran-toxic CryIII proteins, while cryIV genes
encode dipteran-toxic CryIV proteins.
Based on the degree of sequence similarity, the proteins were
further classified into subfamilies; more highly related proteins
within each family were assigned divisional letters such as CryIA,
CryIB, CryIC, etc. Even more closely related proteins within each
division were given names such as CryIC1, CryIC2, etc. Recently a
new nomenclature was developed which systematically classified the
Cry proteins based upon amino acid sequence homology rather than
upon insect target specificities. The classification scheme for
many known toxins, not including allelic variations in individual
proteins, is summarized in Table 1.
TABLE-US-00001 TABLE 1 KNOWN B. THURINGIENSIS .delta.-ENDOTOXINS,
GENBANK ACCESSION NUMBERS, AND REVISED, NOMENCLATURE.sup.A New Old
Genbank Accession # Cry1Aa1 Cry1A(s) M11250 Cry1Aa2 Cry1A(s) M10917
Cry1Aa3 Cry1A(s) D00348 Cry1Aa4 Cry1A(s) X13535 Cry1Aa5 CryIA(a)
D17518 Cry1Aa6 CryIA(a) U43605 Cry1Ab1 CryIA(b) M13898 Cry1Ab2
CryIA(b) M12661 Cry1Ab3 CryIA(b) M15271 Cry1Ab4 CryIA(b) D00117
Cry1Ab5 CryIA(b) X04698 Cry1Ab6 CryIA(b) M37263 Cry1Ab7 CryIA(b)
X13233 Cry1Ab8 CryIA(b) M16463 Cry1Ab9 CryIA(b) X54939 Cry1Ab10
CryIA(b) Cry1Ac1 CryIA(c) M11068 Cry1Ac2 CryIA(c) M35524 Cry1Ac3
CryIA(c) X54159 Cry1Ac4 CryIA(c) M73249 Cry1Ac5 CryIA(c) M73248
Cry1Ac6 CryIA(c) U43606 Cry1Ac7 CryIA(c) U87793 Cry1Ac8 CryIA(c)
U87397 Cry1Ac9 CryIA(c) U89872 Cry1Ac10 CryIA(c) AJ002514 Cry1Ad1
CryIA(d) M73250 Cry1Ae1 CryIA(c) M65252 Cry1Ba1 CryIB X06711
Cry1Ba2 X95704 Cry1Bb1 ET5 L32020 Cry1Bc1 CryIh(c) Z46442 Cry1Bd1
CryEI Cry1Ca1 CryIC X07518 Cry1Ca2 CryIC X13620 Cry1Ca3 CryIC
M73251 Cry1Ca4 CryIC A27642 Cry1Ca5 CryIC X96682 Cry1Co6 CryIC
X96683 Cry1Ca7 CryIC X96684 Cry1Cb1 CryIC(b) M97880 Cry1Da1 CryID
X54160 Cry1Db1 PrtB Z22511 Cry1Ea1 CryIE X53985 Cry1Ea2 CryIE
X56144 Cry1Ea3 CryIE M73252 Cry1Ea4 U94323 Cry1Eb1 CryIE(b) M73253
Cry1Fa1 CryIF M63897 Cry1Fa2 CryIF M63897 Cry1Fb1 PrtD Z22512
Cry1Ga1 PrtA Z22510 Cry1Ga2 CryIM Y09326 Cry1Gb1 CryH2 Cry1Ha1 PrtC
Z22513 Cry1Hb1 U35780 Cry1la1 CryV X62821 Cry1la2 CryV M98544
Cry1la3 CryV L36338 Cry1la4 CryV L49391 Cry1la5 CryV Y08920 Cry1lb1
CryV U07642 Cry1la1 ET4 L32019 Cry1lb1 ET1 U31527 Cry1Ka1 U28801
Cry2Aa1 CryIIA M31738 Cry2Aa2 CryIIA M23723 Cry2Aa3 D86084 Cry2Ab1
CryIIB M23724 Cry2Ab2 CryIIB X55416 Cry2Ac1 CryIIC X57252 Cry3Aa1
CryIIIA M22472 Cry3Aa2 CryIIIA J02978 Cry3Aa3 CryIIIA Y00420
Cry3Aa4 CryIIIA M30503 Cry3Aa5 CryIIIA M37207 Cry3Aa6 CryIIIA
U10985 Cry3Bal CryIIIB X17123 Cry3Ba2 CryIIIB A07234 Cry3Bb1
CryIIIB2 M89794 Cry3Bb2 CryIIIC(b) U31633 Cry3Ca1 CryIIID X59797
Cry4Aa1 CryIVA Y00423 Cry4Aa2 CryIVA D00248 Cry4Ba1 CryIVB X07423
Cry4Ba2 CryIVB X07082 Cry4Ba3 CryIVB M20242 Cry4Ba4 CryIVH D00247
Cry5Aa1 CryVA(a) L07025 Cry5Ab1 CryVA(b) L07026 Cry5Ba1 PS86Q3
U19725 Cry6Aa1 CryVIA L07022 Cry6Ba1 CryVIB L07024 Cry7Aa1 CryIIIC
M64478 Cry7Ab1 CryIIICb U04367 Cry8Aa1 CryIIIE U04364 Cry8Ba1
CryIIIG U04365 Cry8Ca1 CryIIIF U04366 Cry9Aa1 CryIG X58120 Cry9Aa2
CryIG X58534 Cry9Ba1 CryIX X75019 Cry9Ca1 CryIH Z37527 Cry9Da1 N141
D85562 Cry10Aa1 CryIVC M12662 Cry11Aa1 CryIVD M31737 Cry11Aa2
CryIVD M22860 Cry11Ba1 Jeg80 X86902 Cry12Aa1 CryVB L07027 Cry13Aa1
CryVC L07023 Cry14Aa1 CryVD U13955 Cry15Aa1 34kDa M76442 Cry16Aa1
chm71 X94146 Cry17Ao1 cbm71 X99478 Cry18Aa1 CryBP1 X99049 Cry19Aa1
Jeg65 Y08920 Cry20Aa1 U82518 Cry21Aa1 I32932 Cry22Aa1 I34547
Cyt1Aa1 CytA X03182 Cyt1Aa2 CytA X04338 Cyt1Aa3 CytA Y00135 Cyt1Aa4
CytA M35968 Cyt1Ab1 CytM X98793 Cyt1Ba1 U37196 Cyt2Aa1 CytB Z14147
Cyt2Ba1 "CytB" U52043 Cyt2Ba2 "CytB" AF020789 Cyt2Ba3 "CytB"
AF022884 Cyt2Ba4 "CytB" AF022885 Cyt2Ba5 "CytB" AF022886 Cyt2Bb1
U82519 .sup.AAdapted from:
http://epunix.biols.susx.ac.uk/Home/Neil_Grickmore/Bt/index.himl
1.2.3 Bioinsecticide Polypeptide Compositions
The utility of bacterial crystal proteins as insecticides was
extended beyond lepidopterans and dipteran larvae when the first
isolation of a coleopteran-toxic B. thuringiensis strain was
reported (Krieg et al., 1983; 1984). This strain (described in U.S.
Pat. No. 4,766,203, specifically incorporated herein by reference),
designated B. thuringiensis var. tenebrionis, is reported to be
toxic to larvae of the coleopteran insects Agelastica alni (blue
alder leaf beetle) and Leptinotarsa decemlineata (Colorado potato
beetle).
U.S. Pat. No. 5,024, 837 also describes hybrid B. thuringiensis
var. kurstaki strains which showed activity against lepidopteran
insects. U.S. Pat. No. 4,797,279 (corresponding to EP 0221024)
discloses a hybrid B. thuringiensis containing a plasmid from B.
thuringiensis var. kurstaki encoding a lepidopteran-toxic crystal
protein-encoding gene and a plasmid from B. thuringiensis
tenebrionis encoding a coleopteran-toxic crystal protein-encoding
gene. The hybrid B. thuringiensis strain produces crystal proteins
characteristic of those made by both B. thuringiensis kurstaki and
B. thuringiensis tenebrionis. U.S. Pat. No. 4,910,016
(corresponding to EP 0303379) discloses a B. thuringiensis isolate
identified as B. thuringiensis MT 104 which has insecticidal
activity against coleopterans and lepidopterans.
1.2.4 Molecular Genetic Techniques Facilitate Protein
Engineering
The revolution in molecular genetics over the past decade has
facilitated a logical and orderly approach to engineering protein
with improved properties. Site specific and random mutagenesis
methods, the advent of polymerase chain reaction (PCR.TM.)
methodologies, and related advances in the field have permitted an
extensive collection of tools for changing both amino acid
sequence, and underlying genetic sequences for a variety of
proteins of commercial, medical, and agricultural interest.
Following the rapid increase in the number and types of crystal
proteins which have been identified in the past decade, researchers
began to theorize about using such techniques to improve the
insecticidal activity of various crystal proteins. In theory,
improvements to .delta.-endotoxins should be possible using the
methods available to protein engineers working in the art, and it
was logical to assume that it would be possible to isolate improved
variants of the wild-type crystal proteins isolated to date. By
strengthening one or more of the aforementioned steps in the mode
of action of the toxin, improved molecules should provide enhanced
activity, and therefore, represent a breakthrough in the field. If
specific amino acid residues on the protein are identified to be
responsible for a specific step in the mode of action, then these
residues can be targeted for mutagenesis to improve
performance.
1.2.5 Structural Analyses of Crystal Proteins
The combination of structural analyses of B. thuringiensis toxins
followed by an investigation of the function of such structures,
motifs, and the like has taught that specific regions of crystal
protein endotoxins are, in a general way, responsible for
particular functions.
Domain 1, for example, from Cry3Bb and Cry1Ac has been found to be
responsible for ion channel activity, the initial step in formation
of a pore (Walters et al., 1993; Von Tersch et al., 1994). Domains
2 and 3 have been found to be responsible for receptor binding and
insecticidal specificity (Aronson et al., 1995; Caramori et al.,
1991; Chen et al. 1993; de Maagd et al., 1996; Ge et al., 1991; Lee
et al., 1992; Lee et al., 1995; Lu et al., 1994; Smedley and Ellar,
1996; Smith and Ellar, 1994; Rajamohan et al., 1995; Rajamohan et
al., 1996; Wu and Dean, 1996). Regions in domain 2 and 3 can also
impact the ion channel activity of some toxins (Chen et al., 1993,
Wolfersberger et al., 1996; Von Tersch et al., 1994).
1.3 Deficiencies in the Prior Art
Unfortunately, while many laboratories have attempted to make
mutated crystal proteins, few have succeeded in making mutated
crystal proteins with improved lepidopteran toxicity. In almost all
of the examples of genetically-engineered B. thuringiensis toxins
in the literature, the biological activity of the mutated crystal
protein is no better than that of the wild-type protein, and in
many cases, the activity is decreased or destroyed altogether
(Almond and Dean, 1993; Aronson et al., 1995; Chen et al., 1993,
Chen et al., 1995; Ge et al., 1991; Kwak et al., 1995; Lu et al.,
1994; Rajamohan et al., 1995; Rajamohan et al.; 1996; Smedley and
Ellar, 1996; Smith and Ellar, 1994; Wolfersberger et al., 1996; Wu
and Aronson, 1992).
For a crystal protein having approximately 650 amino acids in the
sequence of its active toxin, and the possibility of 20 different
amino acids at each position in this sequence, the likelihood of
arbitrarily creating a successful new structure is remote, even if
a general function to a stretch of 250-300 amino acids can be
assigned. Indeed, the above prior art with respect to crystal
protein gene mutagenesis has been concerned primarily with studying
the structure and function of the crystal proteins, using
mutagenesis to perturb some step in the mode of action, rather than
with engineering improved toxins.
Collectively, the limited successes in the art to develop synthetic
toxins with improved insecticidal activity have stifled progress in
this area and confounded the search for improved endotoxins or
crystal proteins. Rather than following simple and predictable
rules, the successful engineering of an improved crystal protein
may involve different strategies, depending on the crystal protein
being improved and the insect pests being targeted. Thus, the
process is highly empirical.
Accordingly, traditional recombinant DNA technology is clearly not
routine experimentation for providing improved insecticidal crystal
proteins. What are lacking in the prior art are rational methods
for producing genetically-engineered B. thuringiensis crystal
proteins that have improved insecticidal activity and, in
particular, improved toxicity towards a wide range of lepidopteran
insect pests.
2.0 SUMMARY OF THE INVENTION
The present invention seeks to overcome these and other drawbacks
inherent in the prior art by providing genetically-engineered
modified B. thuringiensis .delta.-endotoxins (Cry*), and in
particular modified Cry3 .delta.-endotoxins (designated Cry3*
endotoxins). Also provided are nucleic acid sequences comprising
one or more genes which encode such modified proteins. Particularly
preferred genes include cry3* genes such as cry3A*, cry3B*, and
cry3C* genes, particularly cry3B* genes, and more particularly,
cry3Bb* genes, that encode modified crystal proteins having
improved insecticidal activity against target pests.
Also disclosed are novel methods for constructing synthetic Cry3*
proteins, synthetically-modified nucleic acid sequences encoding
such proteins, and compositions arising therefrom. Also provided
are synthetic cry3* expression vectors and various methods of using
the improved genes and vectors. In a preferred embodiment, the
invention discloses and claims Cry3B* proteins and cry3B* genes
which encode improved insecticidal polypeptides.
In preferred embodiments, channel-forming toxin design methods are
disclosed which have been used to produce a specific set of
designed Cry3Bb* toxins with improved biological activity. These
improved Cry3Bb* proteins are listed in Table 2 along with their
respective amino acid changes from wild-type (WT) Cry3Bb, the
nucleotide changes present in the altered cry3Bb* gene encoding the
protein, the fold increase in bioactivity over WT Cry3Bb, the
structural site of the alteration, and the design method(s) used to
create the new toxins.
Accordingly, the present invention provides in an overall and
general sense, mutagenized Cry3 protein-encoding genes and methods
of making and using such genes. As used herein the term
"mutagenized cry3 gene(s)" means one or more cry3 genes that have
been mutagenized or altered to contain one or more nucleotide
sequences which are not present in the wild type sequences, and
which encode mutant Cry3 crystal proteins (Cry3*) showing improved
insecticidal activity. Such mutagenized cry3 genes have been
referred to in the Specification as cry3* genes. Exemplary cry3*
genes include cry3A*, cry3B*, and cry3C* genes.
Exemplary mutagenized Cry3 protein-encoding genes include cry3B
genes. As used herein the term "mutagenized cry3B gene(s)" means
one or more genes that have been mutagenized or altered to contain
one or more nucleotide sequences which are not present in the wild
type sequences, and which encode mutant Cry3B crystal proteins
(Cry3B*) showing improved insecticidal activity. Such genes have
been designated cry3B* genes. Exemplary cry3B* genes include
cry3Ba* and cry3Bb* genes, which encode Cry3Ba* and Cry3Bb*
proteins, respectively.
Likewise, the present invention provides mutagenized Cry3A
protein-encoding genes and methods of making and using such genes.
As used herein the term "mutagenized cry3A gene(s)" means one or
more genes that have been mutagenized or altered to contain one or
more nucleotide sequences which are not present in the wild type
sequences, and which encode mutant Cry3A crystal proteins (Cry3A*)
showing improved insecticidal activity. Such mutagenized genes have
been designated as cry3A* genes.
In similar fashion, the present invention provides mutagenized
Cry3C protein-encoding genes and methods of making and using such
genes. As used herein the term "mutagenized cry3C gene(s)" means
one or more genes that have been mutagenized or altered to contain
one or more nucleotide sequences which are not present in the wild
type sequences, and which encode mutant Cry3C crystal proteins
(Cry3C*) showing improved insecticidal activity. Such mutagenized
genes have been designated as cry3C* genes.
Preferably the novel sequences comprise nucleic acid sequences in
which at least one, and preferably, more than one, and most
preferably, a significant number, of wild-type cry3 nucleotides
have been replaced with one or more nucleotides, or where one or
more nucleotides have been added to or deleted from the native
nucleotide sequence for the purpose of altering, adding, or
deleting the corresponding amino acids encoded by the nucleic acid
sequence so mutagenized. The desired result, therefore, is
alteration of the amino acid sequence of the encoded crystal
protein to provide toxins having improved or altered activity
and/or specificity compared to that of the unmodified crystal
protein.
Examples of preferred Cry2Bb*-encoding genes include cry3Bb.60,
cry3Bb.11221, cry3Bb.11222, cry3Bb.11223, cry3Bb.11224,
cry3Bb.11225, cry3Bb.11226, cry3Bb.11227, cry3Bb.11228,
cry3Bb.11229, cry3Bb.11230, cry3Bb.11231, cry3Bb.11232,
cry3Bb.11233, cry3Bb.11234, cry3Bb.11235, cry3Bb.11236,
cry3Bb.11237, cry3Bb.11238, cry3Bb.11239, cry3Bb.11241,
cry3Bb.11242, cry3Bb.11032, cry3Bb.11035, cry3Bb.11036,
cry3Bb.11046, cry3Bb.11048, cry3Bb.11051, cry3Bb.11057,
cry3Bb.11058, cry3Bb.11081, cry3Bb.11082, cry3Bb.11083,
cry3Bb.11084, cry3Bb.11095, and cry3Bb.11098.
TABLE-US-00002 TABLE 2 CRY3BB* PROTEINS EXHIBITING IMPROVED
ACTIVITY AGAINST SCRW LARVAE Cry3Bb* cry3Bb* Fold Design Protein
Plasmid cry3Bb* Nucleotide Cry3Bb* Amino Structural Site Increase
Over Method Designation Designation Sequence Changes Acid Changes
of Changes WT Activity Used Cry3Bb.60 -- -- .DELTA.1-159
.DELTA..alpha.1-.alpha.3 3.6x 1, 6, 8 Cry3Bb.11221 pEG1707 A460T,
C461T, A462T, C464A, T154F, P155H, 1.alpha.3, 4 6.4x 1, 8 T465C,
T466C, T467A, A468T, L156H, L158R A469T, G470C, T472C, T473G,
G474T, A477T, A478T, G479C Cry3Bb.11222 pEG1708 T687C, T688C,
A689T, C691A, Y230L, H231S .alpha.6 4.0x 3, 7 A692G Cry3Bb.11223
pEG1709 T667C, T687C, T688A, A689G, S223P, Y230S .alpha.6 2.8x 3
C691A, A692G Cry3Bb.11224 pEG1710 T687C, A692G H231R .alpha.6 5.0x
7, 8 Cry3Bb.11225 pEG1711 T687C, C691A H231N, T241S .alpha.6 3.6x 7
Cry3Rb.11226 pEG1712 T687C, C691A, A692C, T693C H231T .alpha.6 3.0x
7, 8 Cry3Bb.11227 pEG1713 C868A, G869A, G870T R290N 1.alpha.7,
.beta.1 1.9x 2, 3, 4, 6 Cry3Bb.11228 pEG1714 C932T, A938C, T942G,
G949A, S311L, N313T, 1.beta.1, .alpha.8 4.1x 2, 4 T954C E317K
Cry3Bb.11229 pEG1715 T931A, A933C, T942A, T945A, S311T, E317K,
1.beta.1, .alpha.8 2.5x 2, 4 G949A, A953G, T954C Y318C Cry3Bb.11230
pEG1716 T931G, A933C, C934G, T945G, S311A, L312V, 1.beta.1,
.alpha.8 4.7x 2, 4, 8 C946T, A947G, G951A, T954C Q316W Cry3Bb.11231
pEG1717 T687C, A692G, C932T, A938C, H231R, S311L, .alpha.6;
1.beta.1, .alpha.8 7.9x 2, 4, 7, 8, T942G, G949A, T954C N313T,
E317K 10 Cry3Bb.11232 pEG1718 T931A, A933G, T935C, T936A, S311T,
L312P, 1.beta.1, .alpha.8 5.1x 4 A938C, T939C, T942C, T945A, N313T,
E317N G951T, T954C Cry3Bb.11233 pEG1719 T931G, A933C, T936G, T942C,
S311A, Q316D 1.beta.1, .alpha.8 2.2x 2, 4 C943T, T945A, C946G,
G948C, T954C Cry3Bb.11234 pEG1720 T861C, T866C, C868A, T871C,
I289T, L291R, 1.alpha.7, .beta.1 4.1x 4 T872G, A875T, T877A, C878G,
Y292F, S293R A882G Cry3Bb.11235 pEG1721 T687C, A692G, C932T H231R,
S311L .alpha.6; 1.beta.1, .alpha.8 3.2x 2, 4, 7, 8, 10 Cry3Bb.11236
pEG1722 T931A, C932T, A933C, T936C, S311I 1.beta.1, .alpha.8 3.1x
2, 4 T942G, T945A, T954C Cry3Bb.11237 pEG1723 T931A, C932T, A933C,
T936C, S311I, N313H 1.beta.1, .alpha.8 5.4x 2, 4 A937G, A938T,
C941A, T942C, T945A, C946A, A947T, A950T, T954C Cry3Bb.11238
pEG1724 A933C, T936C, A937G, A938T N313V, T314N, 1.beta.1, .alpha.8
2.6x 2, 4 C941A, T942C, T945A, C946A, Q316M, E317V A947T, A950T,
T954C Cry3Bb.11239 pEG1725 A933T, A938G, T939G, T942A, N313R,
L315P, 1.beta.1, .alpha.8 2.8x 2, 4 T944C, T945A, A947T, G948T,
Q316L, E317A A950C, T954C Cry3Bb.11241 pEG1726 A860T, T861C, G862A,
C868T, Y287F, D288N, 1.alpha.7, .beta.1 2.6x 2, 3, 4, 6 G869T,
T871C, A873T, T877A, R290L C878G, A879T Cry3Bb.11242 pEG1727 C868G,
G869T R290V 1.alpha.7, .beta.1 2.5x 2, 3, 4, 6, 8 Cry3Bb.11032
pEG1041 A494G D165G .alpha.4 3.1x 2, 4, 8 Cry3Bb.11035 pEG1046
G479A, A481C, A482C, S160N, K161P, .alpha.4 2.7x 8 A484C, G485A,
A486C, A494G P162H R162H, D165G Cry3Bb.11036 pEG1047 A865G, T877C
I289V, S293P 1.alpha.7, .beta.1 4.3x 4 Cry3Bb.11046 pEG1052 G479A,
A481C, A482C, S160N, K161P, .alpha.4; 1a7, .beta.1 2.6x 2, 4, 8, 10
A484C, G485A, A486C, P162H R162H, A494G, A865G, T877C D165G, I289V,
S293P Cry3Bb.11048 pEG1054 T309A, .DELTA.310, .DELTA.311,
.DELTA.312 D103E, .DELTA.A104 1.alpha.2a, 2b 4.3x 8 Cry3Bb.11051
pEG1057 A565G, A566G K189G 1.alpha.4, 5 3.0x 2, 3, 4 Cry3Bb.11057
pEG1062 T309A, .DELTA.310, .DELTA.311, .DELTA.312, D103E,
.DELTA.A104, 1.alpha.2a, 2b; .alpha.4 3.4x 2, 4, 8, 10 G479A,
A481C, A482C, S160N, K161P, A484C, G485A, A486C, A494G P162H R162H,
D165G Cry3Bb.11058 pEG1063 T309A, .DELTA.310, .DELTA.311,
.DELTA.312, D103E, .DELTA.A104, 1.alpha.2a, 2b; 1.alpha.3, 4 3.5x
1, 8, 10 A460T, C461T, A462T, C464A, T154F, P155H, T465C, T466C,
T467A, A468T, L156H, L158R A469T, G470C, T472C, T473G, G474T,
A477T, A478T, G479C Cry3Bb.11081 pEG1084 A494G, T931A, A933C,
T942A, D165G, S311T, .alpha.4; 1.beta.1, .alpha.8 6.1x 2, 4, 8, 10
T945A, G949A, T954C E317K Cry3Bb.11082 pEG1085 A494G, A865G, T877C,
T914C, D165G, I289V, .alpha.4; 1.alpha.7, .beta.1; .beta.1; 4.9x 2,
4, 5, 8, T931G, A933C, C934G, T945G, S293P, P305S, 1.beta.1,
.alpha.8; .beta.2; 9, 10 C946T, A947G, G951A, T954C, S311A, L312V,
.beta.3b A1043G, T1094C Q316W, Q348R, V365A Cry3Bb.11083 pEG1086
A865G, T877C, A1043G I289V, S293P, 1.alpha.7, .beta.1; .beta.2 7.4x
4, 5, 9, 10 Q348R Cry3Bb.11084 pEG1087 A494G, C932T D165G, S311L
.alpha.4; 1.beta.1, .alpha.8 7.2x 2, 4, 8, 10 Cry3Bb.11095 pEG1095
A1043G Q348R .beta.2 4.6x 5, 9 Cry3Bb.11098 pEG1098 A494G, T687C,
A692G, C932T, D165G, H231R, .alpha.4; .alpha.6, 1.beta.1, .alpha.8
7.9x 2, 4, 7, 8 A938C, T942G, G949A, T954C S311L, N313T, E317K
In a variety of illustrative embodiments, the inventors have shown
remarkable success in generating toxins with improved insecticidal
activity using these methods. In particular, the inventors have
identified unique methods of analyzing and designing toxins having
improved or enhanced insecticidal properties both in vitro and in
vivo.
In addition to modifications of Cry3Bb peptides, those having
benefit of the present teaching are now also able to make mutations
in a variety of channel-forming toxins, and particularly in crystal
proteins which are related to Cry3Bb either functionally or
structurally. In fact, the inventors contemplate that any B.
thuringiensis crystal protein or peptide can be analyzed using the
methods disclosed herein and may be altered using the methods
disclosed herein to produce crystal proteins having improved
insecticidal specificity or activity. Alternatively, the inventors
contemplate that those of skill in the art having the benefit of
the teachings disclosed herein will be able to prepare not only
mutated Cry3 toxins with improved activity, but also other crystal
proteins including all of those proteins identified in Table 1,
herein. In particular, the inventors contemplate the creation of
Cry3* variants using one or more of the methods disclosed herein to
produce toxins with improved activity. For example, the inventors
note Cry3A, Cry3B, and Cry3C crystal proteins (which are known in
the art) may be modified using one or more of the design strategies
employed herein, to prepare synthetically-modifiedcrystal proteins
with improved properties. Likewise, one of skill in the art will
even be able to utilize the teachings of the present disclosure to
modify other channel forming toxins, including channel forming
toxins other than B. thuringiensis crystal proteins, and even to
modify proteins and channel toxins not yet described or
characterized.
Because the structures for insecticidal crystal proteins show a
remarkable conservation of protein tertiary structure (Grochulski
et al., 1995), and because many crystal proteins show significant
amino acid sequence identity to the Cry3Bb amino acid sequence
within domain 1, including proteins of the Cry1, Cry2, Cry3, Cry4,
Cry5, Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, and
Cry16 classes (Table 1), now in light of the inventors' surprising
discovery, for the first time, those of skill in the art having
benefit of the teachings disclosed herein will be able to broadly
apply the methods of the invention to modifying a host of crystal
proteins with improved activity or altered specificity. Such
methods will not only be limited to the insecticidal crystal
proteins disclosed in Table 1, but may also been applied to any
other related crystal protein, including those yet to be
identified.
In particular, the high degree of homology between Cry3A, Cry3B,
and Cry3C proteins is evident in the alignment of the primary amino
acid sequence of the three proteins (FIG. 17A, FIG. 17B, and FIG.
17C).
As such, the disclosed methods may be now applied to preparation of
modified crystal proteins having one or more alterations introduced
using one or more of the mutational design methods as disclosed
herein. The inventors further contemplate that regions may be
identified in one or more domains of a crystal protein, or other
channel forming toxin which may be similarly modified through
site-specific or random mutagenesis to generate toxins having
improved activity, or alternatively, altered specificity.
In certain applications, the creation of altered toxins having
increased activity against one or more insects is desired.
Alternatively, it may be desirable to utilize the methods described
herein for creating and identifying altered insecticidal crystal
proteins which are active against a wider spectrum of susceptible
insects. The inventors further contemplate that the creation of
chimeric insecticidal crystal proteins comprising one or more of
these mutations may be desirable for preparing "super" toxins which
have the combined advantages of increased insecticidal activity and
concomitant broad spectrum activity.
In light of the present disclosure, the mutagenesis of one or more
codons within the sequence of a toxin may result in the generation
of a host of related insecticidal proteins having improved
activity. While exemplary mutations have been described for each of
the design strategies employed in the present invention, the
inventors contemplate that mutations may also be made in
insecticidal crystal proteins, including the loop regions, helices
regions, active sites of the toxins, regions involved in protein
oligomerization, and the like, which will give rise to functional
bioinsecticidal crystal proteins. All such mutations are considered
to fall within the scope of this disclosure.
In one illustrative embodiment, mutagenized cry3Bb* genes are
obtained which encode Cry3Bb* variants that are generally based
upon the wild-type Cry3Bb sequence, but that have one or more
changes incorporated into the amino acid sequence of the protein
using one or more of the design strategies described and claimed
herein.
In these and other embodiments, the mutated genes encoding the
crystal proteins may be modified so as to change about one, two,
three, four, or five or so amino acids in the primary sequence of
the encoded polypeptide. Alternatively even more changes from the
native sequence may be introduced, such that the encoded protein
may have at least about 1% or 2%, or alternatively about 3% or
about 4%, or even about 5% to about 10%, or about 10% to about 15%,
or even about 15% to about 20% or more of the codons either
altered, deleted, or otherwise modified. In certain situations, it
may even be desirable to alter substantially more of the primary
amino acid sequence to obtain the desired modified protein. In such
cases the inventors contemplate that from about 25%, to about 50%,
or even from about 50% to about 75%, or more of the native (or
wild-type) codons either altered, deleted, or otherwise modified.
Alternatively, mutations in the amino acid sequences or underlying
DNA gene sequences which result in the insertion or deletion of one
or more amino acids within one or more regions of the crystal
protein or peptide.
To effect such changes in the primary sequence of the encoded
polypeptides, it may be desirable to mutate or delete one or more
nucleotides from the nucleic acid sequences of the genes encoding
such polypeptides, or alternatively, under certain circumstances to
add one or more nucleotides into the primary nucleic acid sequence
at one or more sites in the sequence. Frequently, several
nucleotide residues may be altered to produce the desired
polypeptide. As such, the inventors contemplate that in certain
embodiments it may be desirable to alter only one, two, three,
four, or five or so nucleotides in the primary sequence. In other
embodiments, which more changes are desired, the mutagenesis may
involve changing, deleting, or inserting 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or even 20 or so nucleotide residues in
the gene sequence. In still other embodiments, one may desire to
mutate, delete, or insert 21, 22, 23, 24, 25, 26, 27, 28, 29,
30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or even 90-100, 150, 200,
250, 300, 350, 400, 450, or more nucleotides in the sequence of the
gene in order to prepare a cry3* gene which produces a Cry3*
polypeptide having the desired characteristics. In fact, any number
of mutations, deletions, and/or insertions may be made in the
primary sequence of the gene, so long as the encoded protein has
the improved insecticidal activity or specificity characteristics
described herein.
Changing a large number of the codons in the nucleotide sequence of
an endotoxin-encoding gene may be particularly desirable and often
necessary to achieve the desired results, particularly in the
situation of "plantizing" a DNA sequence in order to express a DNA
of non-plant origin in a transformed plant cell. Such methods are
routine to those of skill in the plant genetics arts, and
frequently many residues of a primary gene sequence will be altered
to facilitate expression of the gene in the plant cell. Preferably,
the changes in the gene sequence introduce no changes in the amino
acid sequence, or introduce only conservative replacements in the
amino acid sequence such that the polypeptide produced in the plant
cell from the "plantized" nucleotide sequence is still fully
functional, and has the desired qualities when expressed in the
plant cell.
Genes and encoded proteins mutated in the manner of the invention
may also be operatively linked to other protein-encoding nucleic
acid sequences, or expressed as fusion proteins. Both N-terminal
and C-terminal fusion proteins are contemplated. Virtually any
protein- or peptide-encoding DNA sequence, or combinations thereof,
may be fused to a mutated cry3* sequence in order to encode a
fusion protein. This includes DNA sequences that encode targeting
peptides, proteins for recombinant expression, proteins to which
one or more targeting peptides is attached, protein subunits,
domains from one or more crystal proteins, and the like. Such
modifications to primary nucleotide sequences to enhance, target,
or optimize expression to the gene sequence in a particular host
cell, tissue, or cellular localization, are well-known to those of
skill in the art of protein engineering and molecular biology, and
it will be readily apparent to such artisans, having benefit of the
teachings of this specification, how to facilitate such changes in
the nucleotide sequence to produce the polypeptides and
polynucleotides disclosed herein.
In one aspect, the invention discloses and claims host cells
comprising one or more of the modified crystal proteins disclosed
herein, and in particular, cells of B. thuringiensis strains
EG11221, EG11222, EG11223, EG11224, EG11225, EG11226, EG11227,
EG11228, EG11229, EG11230, EG11231, EG11232, EG11233, EG11234,
EG11235, EG11236, EG11237, EG11238, EG11239, EG11241, EG11242,
EG11032, EG11035, EG11036, EG11046, EG11048, EG11051, EG11057,
EG11058, EG11081, EG11082, EG11083, EG11084, EG11095, and EG11098
which comprise recombinant DNA segments encoding
synthetically-modified Cry3Bb* crystal proteins which demonstrates
improved insecticidal activity.
Likewise, the invention also discloses and claims cell cultures of
B. thuringiensis EG11221, EG11222, EG11223, EG11224, EG11225,
EG11226, EG11227, EG11228, EG11229, EG11230, EG11231, EG11232,
EG11233, EG11234, EG11235, EG11236, EG11237, EG11238, EG11239,
EG11241, EG11242, EG11032, EG11035, EG11036, EG11046, EG11048,
EG11051, EG11057, EG11058, EG11081, EG11082, EG11083, EG11084, and
EG11095, and EG11098.
Such cell cultures may be biologically-pure cultures consisting of
a single strain, or alternatively may be cell co-cultures
consisting of one or more strains. Such cell cultures may be
cultivated under conditions in which one or more additional B.
thuringiensis or other bacterial strains are simultaneously
co-cultured with one or more of the disclosed cultures, or
alternatively, one or more of the cell cultures of the present
invention may be combined with one or more additional B.
thuringiensis or other bacterial strains following the independent
culture of each. Such procedures may be useful when suspensions of
cells containing two or more different crystal proteins are
desired.
The subject cultures have been deposited under conditions that
assure that access to the cultures will be available during the
pendency of this patent application to one determined by the
Commissioner of Patents and Trademarks to be entitled thereto under
37 C.F.R. .sctn.1.14 and 35 U.S.C. .sctn.122. The deposits are
available as required by foreign patent laws in countries wherein
counterparts of the subject application, or its progeny, are filed.
However, it should be understood that the availability of a deposit
does not constitute a license to practice the subject invention in
derogation of patent rights granted by governmental action.
Further, the subject culture deposits will be stored and made
available to the public in accord with the provisions of the
Budapest Treaty for the Deposit of Microorganisms, i.e., they will
be stored with all the care necessary to keep them viable and
uncontaminated for a period of at least five years after the most
recent request for the finishing of a sample of the deposit, and in
any case, for a period of at least 30 (thirty) years after the date
of deposit or for the enforceable life of any patent which may
issue disclosing the cultures. The depositor acknowledges the duty
to replace the deposits should the depository be unable to furnish
a sample when requested, due to the condition of the deposits. All
restrictions on the availability to the public of the subject
culture deposits will be irrevocably removed upon the granting of a
patent disclosing them.
Cultures shown in Table 3 were deposited in the permanent
collection of the Agricultural Research Service Culture Collection,
Northern Regional Research Laboratory (NRRL) under the terms of the
Budapest Treaty.
TABLE-US-00003 TABLE 3 STRAINS OF THE PRESENT INVENTION DEPOSITED
UNDER THE TERMS OF THE BUDAPEST TREATY Accession Number Strain
Deposit Date Protein (NRRL Number) EG11032 5/27/97 Cry3Bb.11032
B-21744 EG11035 5/27/97 Cry3Bb.11035 B-21745 EG11036 5/27/97
Cry3Bb.11036 B-21746 EG11037 5/27/97 Cry3Bb.11037 B-21747 EG11046
5/27/97 Cry3Bb.11046 B-21748 EG11048 5/27/97 Cry3Bb.11048 B-21749
EG11051 5/27/97 Cry3Bb.11051 B-21750 EG11057 5/27/97 Cry3Bb.11057
B-21751 EG11058 5/27/97 Cry3Bb.11058 B-21752 EG11081 5/27/97
Cry3Bb.11081 B-21753 EG11082 5/27/97 Cry3Bb.11082 B-21754 EG11083
5/27/97 Cry3Bb.11083 B-21755 EG11084 5/27/97 Cry3Bb.11084 B-21756
EG11095 5/27/97 Cry3Bb.11095 B-21757 EG11204 5/27/97 Cry3Bb.11204
B-21758 EG11221 5/27/97 Cry3Bb.11221 B-21759 EG11222 5/27/97
Cry3Bb.11222 B-21760 EG11223 5/27/97 Cry3Bb.11223 B-21761 EG11224
5/27/97 Cry3Bb.11224 B-21762 EG11225 5/27/97 Cry3Bb.11225 B-21763
EG11226 5/27/97 Cry3Bb.11226 B-21764 EG11227 5/27/97 Cry3Bb.11227
B-12765 EG11228 5/27/97 Cry3Bb.11228 B-12766 EG11229 5/27/97
Cry3Bb.11229 B-21767 EG11230 5/27/97 Cry3Bb.11230 B-21768 EG11231
5/27/97 Cry3Bb.11231 B-21769 EG11232 5/27/97 Cry3Bb.11232 B-12770
EG11133 5/27/97 Cry3Bb.11233 B-21771 EG11234 5/27/97 Cry3Bb.11234
B-21772 EG11235 5/27/97 Cry3Bb.11235 B-21773 EG11236 5/27/97
Cry3Bb.11236 B-21774 EG11237 5/27/97 Cry3Bb.11237 B-21775 EG11238
5/27/97 Cry3Bb.11238 B-21776 EG11239 5/27/97 Cry3Bb.11239 B-21777
EG11241 5/27/97 Cry3Bb.11241 B-21778 EG11242 5/27/97 Cry3Bb.11242
B-21779
Also disclosed are methods of controlling or eradicating an insect
population from an environment. Such methods generally comprise
contacting the insect population to be controlled or eradicated
with an insecticidally-effective amount of a Cry3* crystal protein
composition. Preferred Cry3* compositions include Cry3A*, Cry3B*,
and Cry3C* polypeptide compositions, with Cry3B* compositions being
particularly preferred. Examples of such polypeptides include
proteins selected from the group consisting of Cry3Bb-60,
Cry3Bb.11221, Cry3Bb.11222, Cry3Bb.11223, Cry3Bb.11224,
Cry3Bb.11225, Cry3Bb.11226, Cry3Bb.11227, Cry3Bb.11228,
Cry3Bb.11229, Cry3Bb.11230, Cry3Bb.11231, Cry3Bb.11232,
Cry3Bb.11233, Cry3Bb.11234, Cry3Bb.11235, Cry3Bb.11236,
Cry3Bb.11237, Cry3Bb.11238, Cry3Bb.11239, Cry3Bb.11241,
Cry3Bb.11242, Cry3Bb.11032, Cry3Bb.11035, Cry3Bb.11036,
Cry3Bb.11046, Cry3Bb.11048, Cry3Bb.11051, Cry3Bb.11057,
Cry3Bb.11058, Cry3Bb.11081, Cry3Bb.11082, Cry3Bb.11083,
Cry3Bb.11084, Cry3Bb.11095, and Cry3Bb.11098.
In preferred embodiments, these Cry3Bb* crystal protein
compositions comprise the amino acid sequence of any of SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6. SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:14. SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ
ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30,
SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID
NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ
ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58,
SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID
NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ ID NO:102 or SEQ ID
NO:108.
2.1 Methods for Producing Modified Cry* Proteins
The modified Cry* polypeptides of the present invention are
preparable by a process which generally involves the steps of
obtaining a nucleic acid sequence encoding a Cry* polypeptide;
analyzing the structure of the polypeptide to identify particular
"target" sites for mutagenesis of the underlying gene sequence;
introducing one or more mutations into the nucleic acid sequence to
produce a change in one or more amino acid residues in the encoded
polypeptide sequence; and expressing in a transformed host cell the
mutagenized nucleic acid sequence under conditions effective to
obtain the modified Cry* protein encoded by the cry* gene.
Means for obtaining the crystal structures of the polypeptides of
the invention are well-known. Exemplary high resolution crystal
structure solution sets are given in Section 9.0 of the disclosure,
and include the crystal structure of both the Cry3A and Cry3B
polypeptides disclosed herein. The information provided in Section
9.0 permits the analyses disclosed in each of the methods herein
which rely on the 3D crystal structure information for targeting
mutagenesis of the polypeptides to particular regions of the
primary amino acid sequences of the .delta.-endotoxins to obtain
mutants with increased insecticidal activity or enhanced
insecticidal specificity.
A first method for producing a modified B. thuringiensis Cry3Bb
.delta.-endotoxin having improved insecticidal activity or
specificity disclosed herein generally involves obtaining a
high-resolution 3D crystal structure of the endotoxin, locating in
the crystal structure one or more regions of bound water wherein
the bound water forms a contiguous hydrated surfaces separated by
no more than about 16 .ANG.; increasing the number of water
molecules in this surface by increasing the hydrophobicity of one
or more amino acids of the protein in the region; and obtaining the
modified .delta.-endotoxin so produced. Exemplary
.delta.-endotoxins include Cry3Bb.11032, Cry3Bb.11227,
Cry3Bb.11241, Cry3Bb.11051, Cry3Bb.11242, and Cry3Bb.11098.
A second method for producing a modified B. thuringiensis Cry3Bb
.delta.-endotoxin having improved insecticidal activity comprises
identifying a loop region in a .delta.-endotoxin; modifying one or
more amino acids in the loop to increase the hydrophobicity of the
amino acids; and obtaining the modified .delta.-endotoxin so
produced. Preferred .delta.-endotoxin-produced by this method
include Cry3Bb.11241, Cry3Bb.11242, Cry3Bb.11228, Cry3Bb.11229,
Cry3Bb.11230, Cry3Bb.11231, Cry3Bb.11233, Cry3Bb.11236,
Cry3Bb.11237, Cry3Bb.11238, and Cry3Bb.11239.
A method for increasing the mobility of channel forming helices of
a B. thuringiensis Cry3B .delta.-endotoxin is also provided by the
present invention. The method generally comprises disrupting one or
more hydrogen bonds formed between a first amino acid of one or
more of the channel forming helices and a second amino acid of the
.delta.-endotoxin. The hydrogen bonds may be formed inter- or
intramolecularly, and the disrupting may consist of replacing a
first or second amino acid with a third amino acid whose spatial
distance is greater than about 3 .ANG., or whose spatial
orientation bond angle is not equal to 180.+-.60 degrees relative
to the hydrogen bonding site of the first or second amino acid.
.delta.-endotoxins produced by this method and disclosed herein
include Cry3Bb.11222, Cry3Bb.11223, Cry3Bb.11224, Cry3Bb.11225,
Cry3Bb.11226, Cry3Bb.11227, Cry3Bb.11231, Cry3Bb.11241, and
Cry3Bb.11242, and Cry3Bb.11098.
Also disclosed is a method of increasing the flexibility of a loop
region in a channel forming domain of a B. thuringiensis Cry3Bb
.delta.-endotoxin. This method comprises obtaining a crystal
structure of a Cry3Bb .delta.-endotoxin having one or more loop
regions; identifying the amino acids comprising the loop region;
and altering one or more of the amino acids to reduce steric
hindrance in the loop region, wherein the altering increases
flexibility of the loop region in the .delta.-endotoxin. Examples
of .delta.-endotoxins produced using this method include
Cry3Bb.11032, Cry3Bb.11051, Cry3Bb.11228, Cry3Bb.11229,
Cry3Bb.11230, Cry3Bb.11231, Cry3Bb.11232, Cry3Bb.11233,
Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238, Cry3Bb.11239,
Cry3Bb.11227, Cry3Bb.11234, Cry3Bb.11241, Cry3Bb.11242,
Cry3Bb.11036, and Cry3Bb.11098.
Another aspect of the invention is a method for increasing the
activity of a .delta.-endotoxin, comprising reducing or eliminating
binding of the .delta.-endotoxin to a carbohydrate in a target
insect gut. The eliminating or reducing may be accomplished by
removal of one or more a helices of domain 1 of the
.delta.-endotoxin, for example, by removal of .alpha. helices
.alpha.1, .alpha.2a/b, and .alpha.3. An exemplary .delta.-endotoxin
produced using the method is Cry3Bb.60.
Alternatively, the reducing or eliminating may be accomplished by
replacing one or more amino acids within loop Pl,o8, with one or
more amino acids having increased hydrophobicity. Such a method
gives rise to .delta.-endotoxins such as Cry3Bb.11228,
Cry3Bb.11230, Cry3Bb.11231, Cry3Bb.11237, and Cry3Bb.11098, which
are described in detail, herein.
Alternatively, the reducing or eliminating is accomplished by
replacing one or more specific amino acids, with any other amino
acid. Such replacements are described in Table 2, and in the
examples herein. One example is the .delta.-endotoxin designated
herein as Cry3Bb.11221.
A method of identifying a region of a Cry3Bb .delta.-endotoxin for
targeted mutagenesis comprising: obtaining a crystal structure of
the .delta.-endotoxin; identifying from the crystal structure one
or more surface-exposed amino acids in the protein; randomly
substituting one or more of the surface-exposed amino acids to
obtain a plurality of mutated polypeptides, wherein at least 50% of
the mutated polypeptides have diminished insecticidal activity; and
identifying from the plurality of mutated polypeptides one or more
regions of the Cry3Bb .delta.-endotoxin for targeted mutagenesis.
The method may further comprise determining the amino acid
sequences of a plurality of mutated polypeptides having diminished
activity, and identifying one or more amino acid residues required
for insecticidal activity.
In another embodiment, the invention provides a process for
producing a Cry3Bb .delta.-endotoxin having improved insecticidal
activity. The process generally involves the steps of obtaining a
high-resolution crystal structure of the protein; determining the
electrostatic surface distribution of the protein; identifying one
or more regions of high electrostatic diversity; modifying the
electrostatic diversity of the region by altering one or more amino
acids in the region; and obtaining a Cry3Bb .delta.-endotoxin which
has improved insecticidal activity. In one embodiment, the
electrostatic diversity may be decreased relative to the
electrostatic diversity of a native Cry3Bb .delta.-endotoxin.
Exemplary .delta.-endotoxins with decreased electrostatic diversity
include Cry3Bb.11227, Cry3Bb.11241, and Cry3Bb.11242.
Alternatively, the electrostatic diversity may be increased
relative to the electrostatic diversity of a native Cry3Bb
.delta.-endotoxin. An exemplary .delta.-endotoxin with increased
electrostatic diversity is Cry3Bb.11234.
Furthermore, the invention also provides a method of producing a
Cry3Bb .delta.-endotoxin having improved insecticidal activity
which involves obtaining a high-resolution crystal structure;
identifying the presence of one or more metal binding sites in the
protein; altering one or more amino acids in the binding site; and
obtaining an altered protein, wherein the protein has improved
insecticidal activity. The altering may involve the elimination of
one or more metal binding sites. Exemplary .delta.-endotoxin
include Cry3Bb.11222, Cry3Bb.11224, Cry3Bb.11225, and
Cry3Bb.11226.
A further aspect of the invention involves a method of identifying
a B. thuringiensis Cry3Bb .delta.-endotoxin having improved channel
activity. This method in an overall sense involves obtaining a
Cry3Bb .delta.-endotoxin suspected of having improved channel
activity; and determining one or more of the following
characteristics in the .delta.-endotoxin, and comparing such
characteristics to those obtained for the wild-type unmodified
.delta.-endotoxin: (1) the rate of channel formation, (2) the rate
of growth of channel conductance or (3) the duration of open
channel state. From this comparison, one may then select a
.delta.-endotoxin which has an increased rate of channel formation
compared to the wild-type .delta.-endotoxin. Examples of Cry3Bb
.delta.-endotoxins prepared by this method include Cry3Bb.60,
Cry3Bb.11035, Cry3Bb.11048, Cry3Bb.11032, Cry3Bb.11223,
Cry3Bb.11224, Cry3Bb.11226, Cry3Bb.11221, Cry3Bb.11242,
Cry3Bb.11230, and Cry3Bb.11098.
Also provided is a method for producing a modified Cry3Bb
.delta.-endotoxin, having improved insecticidal activity which
involves altering one or more non-surface amino acids located at or
near the point of greatest convergence of two or more loop regions
of the Cry3Bb .delta.-endotoxin, such that the altering decreases
the mobility of one or more of the loop regions. The mobility may
conveniently be determined by comparing the thermal denaturation of
the modified protein to a wild-type Cry3Bb .delta.-endotoxin. An
exemplary crystal protein produced by this method is
Cry3Bb.11095.
A further aspect of the invention involves a method for preparing a
modified Cry3Bb .delta.-endotoxin, having improved insecticidal
activity comprising modifying one or more amino acids in the loop
to increase the hydrophobicity of said amino acids; and altering
one or more of said amino acids to reduce steric hindrance in the
loop region, wherein the altering increases flexibility of the loop
region in the endotoxin. Exemplary Cry3Bb .delta.-endotoxins
produced is selected from the group consisting of Cry3Bb.11057,
Cry3Bb.11058, Cry3Bb.11081, Cry3Bb.11082, Cry3Bb.11083,
Cry3Bb.11084, Cry3Bb.11231, Cry3Bb.11235, and Cry3Bb.11098.
The invention also provides a method of improving the insecticidal
activity of a B. thuringiensis Cry3Bb .delta.-endotoxin, which
generally comprises inserting one or more protease sensitive sites
into one or more loop regions of domain 1 of the .delta.-endotoxin.
Preferably, the loop region is .alpha.3,4, and an exemplary
.delta.-endotoxin so produced is Cry3Bb.11221.
2.2 Polypeptide Compositions
The crystal proteins so produced by each of the methods described
herein also represent important aspects of the invention. Such
crystal proteins preferably include a protein or peptide selected
from the group consisting of Cry3Bb-60, Cry3Bb.11221, Cry3Bb.11222,
Cry3Bb.11223, Cry3Bb.11224, Cry3Bb.11225, Cry3Bb.11226,
Cry3Bb.11227, Cry3Bb.11228, Cry3Bb.11229, Cry3Bb.11230,
Cry3Bb.11231, Cry3Bb.11232, Cry3Bb.11233, Cry3Bb.11234,
Cry3Bb.11235, Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238,
Cry3Bb.11239, Cry3Bb.11241, Cry3Bb.11242, Cry3Bb.11032,
Cry3Bb.11035, Cry3Bb.11036, Cry3Bb.11046, Cry3Bb.11048,
Cry3Bb.11051, Cry3Bb.11057, Cry3Bb.11058, Cry3Bb.11081,
Cry3Bb.11082, Cry3Bb.11083, Cry3Bb.11084, Cry3Bb.11095, and
Cry3Bb.11098.
In preferred embodiments, the protein comprises a contiguous amino
acid sequence selected from the group consisting of SEQ ID NO:2,
SEQ ID NO:4, SEQ ID NO:6. SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,
SEQ ID NO:14. SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID
NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ
ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40,
SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID
NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ
ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68,
SEQ ID NO:70, SEQ ID NO:100, SEQ ID NO:102, and SEQ ID NO:108.
Highly preferred are those crystal proteins which are encoded by
the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:5. SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13. SEQ ID
NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ
ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33,
SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID
NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ
ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61,
SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID
NO:99, SEQ ID NO:101; or SEQ ID NO:107, or a nucleic acid sequence
which hybridizes to the nucleic acid sequence of SEQ ID NO:1, SEQ
ID NO:3, SEQ ID NO:5. SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ
ID NO:13. SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,
SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID
NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ
ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49,
SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID
NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ
ID NO:69, SEQ ID NO:99, SEQ ID NO:101, or SEQ ID NO:107 under
conditions of moderate stringency.
Amino acid, peptide and protein sequences within the scope of the
present invention include, and are not limited to the sequences set
forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ
ID NO:20, SEQ ID NO:22 SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28,
SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID
NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 SEQ
ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56,
SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID
NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ ID NO:102,
and SEQ ID NO:108, and alterations in the amino acid sequences
including alterations, deletions, mutations, and homologs.
Compositions which comprise from about 0.5% to about 99% by weight
of the crystal protein, or more preferably from about 5% to about
75%, or from about 25% to about 50% by weight of the crystal
protein are provided herein. Such compositions may readily be
prepared using techniques of protein production and purification
well-known to those of skill, and the methods disclosed herein.
Such a process for preparing a Cry3Bb* crystal protein generally
involves the steps of culturing a host cell which expresses the
Cry3Bb* protein (such as a B. thuringiensis EG11221, EG11222,
EG11223, EG11224, EG11225, EG11226, EG11227, EG11228, EG11229,
EG11230, EG11231, EG11232, EG11233, EG11234, EG11235, EG11236,
EG11237, EG11238, EG11239, EG11241, EG11242, EG11032, EG11035,
EG11036, EG11046, EG11048, EG11051, EG11057, EG11058, EG11081,
EG11082, EG11083, EG11084, EG11095, or EG11098 cell) under
conditions effective to produce the crystal protein, and then
obtaining the crystal protein so produced.
The protein may be present within intact cells, and as such, no
subsequent protein isolation or purification steps may be required.
Alternatively, the cells may be broken, sonicated, lysed,
disrupted, or plasmolyzed to free the crystal protein(s) from the
remaining cell debris. In such cases, one may desire to isolate,
concentrate, or further purify the resulting crystals containing
the proteins prior to use, such as, for example, in the formulation
of insecticidal compositions. The composition may ultimately be
purified to consist almost entirely of the pure protein, or
alternatively, be purified or isolated to a degree such that the
composition comprises the crystal protein(s) in an amount of from
between about 0.5% and about 99% by weight, or in an amount of from
between about 5% and about 95% by weight, or in an amount of from
between about 15% and about 85% by weight, or in an amount of from
between about 25% and about 75% by weight, or in an amount of from
between about 40% and about 60% by weight etc.
2.3 Recombinant Vectors Expressing Cry3* Genes
One important embodiment of the invention is a recombinant vector
which comprises a nucleic acid segment encoding one or more of the
novel B. thuringiensis crystal proteins disclosed herein. Such a
vector may be transferred to and replicated in a prokaryotic or
eukaryotic host, with bacterial cells being particularly preferred
as prokaryotic hosts, and plant cells being particularly preferred
as eukaryotic hosts.
In preferred embodiments, the recombinant vector comprises a
nucleic acid segment encoding the amino acid sequence of SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ
ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30,
SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID
NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ
ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58,
SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID
NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ ID NO:102, or SEQ ID
NO:108. Highly preferred nucleic acid segments are those which have
the sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ
ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35,
SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID
NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ
ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63,
SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ ID
NO:101, or SEQ ID NO:107.
Another important embodiment of the invention is a transformed host
cell which expresses one or more of these recombinant vectors. The
host cell may be either prokaryotic or eukaryotic, and particularly
preferred host cells are those which express the nucleic acid
segment(s) comprising the recombinant vector which encode one or
more B. thuringiensis crystal protein comprising modified amino
acid sequences in one or more loop regions of domain 1, or between
.alpha. helix 7 of domain 1 and .beta. strand 1 of domain 2.
Bacterial cells are particularly preferred as prokaryotic hosts,
and plant cells are particularly preferred as eukaryotic hosts
In an important embodiment, the invention discloses and claims a
host cell wherein the modified amino acid sequences comprise one or
more loop regions between .alpha. helices 1 and 2, .alpha. helices
2 and 3, .alpha. helices 3 and 4, .alpha. helices 4 and 5, .alpha.
helices 5 and 6 or .alpha. helices 6 and 7 of domain 1, or between
.alpha. helix 7 of domain 1 and .beta. strand 1 of domain 2. A
particularly preferred host cell is one that comprises the amino
acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID
NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ
ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26,
SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID
NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ
ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54,
SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID
NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ
ID NO:102, or SEQ ID NO:108, and more preferably, one that
comprises the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3,
SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,
SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID
NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ
ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41,
SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID
NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ
ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69,
SEQ ID NO:99, SEQ ID NO:101, or SEQ ID NO:107.
Bacterial host cells transformed with a nucleic acid segment
encoding a modified Cry3Bb crystal protein according to the present
invention are disclosed and claimed herein, and in particular, a B.
thuringiensis cell having designation EG11221, EG11222, EG11223,
EG11224, EG11225, EG11226, EG11227, EG11228, EG11229, EG11230,
EG11231, EG11232, EG11233, EG11234, EG11235, EG11236, EG11237,
EG11238, EG11239, EG11241, EG11242, EG11032, EG11035, EG11036,
EG11046, EG11048, EG11051, EG11057, EG11058, EG11081, EG11082,
EG11083, EG11084, EG11095, or EG11098.
In another embodiment, the invention encompasses a method of using
a nucleic acid segment of the present invention that encodes a
cry3Bb* gene. The method generally comprises the steps of: (a)
preparing a recombinant vector in which the cry3Bb* gene is
positioned under the control of a promoter; (b) intoducing the
recombinant vector into a host cell; (c) culturing the host cell
under conditions effective to allow expression of the Cry3Bb*
crystal protein encoded by said cry3Bb* gene; and (d) obtaining the
expressed Cry3Bb* crystal protein or peptide.
A wide variety of ways are available for introducing a B.
thuringiensis gene expressing a toxin into the microorganism host
under conditions which allow for stable maintenance and expression
of the gene. One can provide for DNA constructs which include the
transcriptional and translational regulatory signals for expression
of the toxin gene, the toxin gene under their regulatory control
and a DNA sequence homologous with a sequence in the host oganism,
whereby integration will occur, and/or a replication system which
is functional in the host, whereby integration or stable
maintenance will occur.
The transcriptional initiation signals will include a promoter and
a transcriptional initiation start site. In some instances, it may
be desirable to provide for regulative expression of the toxin,
where expression of the toxin will only occur after release into
the environment. This can be achieved with operators or a region
binding to an activator or enhancers, which are capable of
induction upon a change in the physical or chemical environment of
the microorganisms. For example, a temperature sensitive regulatory
region may be employed, where the organisms may be grown up in the
laboratory without expression of a toxin, but upon release into the
environment, expression would begin. Other techniques may employ a
specific nutrient medium in the laboratory, which inhibits the
expression of the toxin, where the nutrient medium in the
environment would allow for expression of the toxin. For
translational initiation, a ribosomal binding site and an
initiation codon will be present.
Various manipulations may be employed for enhancing the expression
of the messenger RNA, particularly by using an active promoter, as
well as by employing sequences, which enhance the stability of the
messenger RNA. The transcriptional and translational termination
region will involve stop codon(s), a terminator region, and
optionally, a polyadenylation signal. A hydrophobic "leader"
sequence may be employed at the amino terminus of the translated
polypeptide sequence in order to promote secretion of the protein
across the inner membrane.
In the direction of transcription, namely in the 5' to 3' direction
of the coding or sense sequence, the construct will involve the
transcriptional regulatory region, if any, and the promoter, where
the regulatory region may be either 5' or 3' of the promoter, the
ribosomal binding site, the initiation codon, the structural gene
having an open reading frame in phase with the initiation codon,
the stop codon(s), the polyadenylation signal sequence, if any, and
the terminator region. This sequence as a double strand may be used
by itself for transformation of a microorganism host, but will
usually be included with a DNA sequence involving a marker, where
the second DNA sequence may be joined to the toxin expression
construct during introduction of the DNA into the host.
By a marker is intended a structural gene which provides for
selection of those hosts which have been modified or transformed.
The marker will normally provide for selective advantage, for
example, providing for biocide resistance, e.g., resistance to
antibiotics or heavy metals; complementation, so as to provide
prototropy to an auxotrophic host, or the like. Preferably,
complementation is employed, so that the modified host may not only
be selected, but may also be competitive in the field. One or more
markers may be employed in the development of the constructs, as
well as for modifying the host. The organisms may be further
modified by providing for a competitive advantage against other
wild-type microorganisms in the field. For example, genes
expressing metal chelating agents, e.g., siderophores, may be
introduced into the host along with the structural gene expressing
the toxin. In this manner, the enhanced expression of a siderophore
may provide for a competitive advantage for the toxin-producing
host, so that it may effectively compete with the wild-type
microorganisms and stably occupy a niche in the environment.
Where no functional replication system is present, the construct
will also include a sequence of at least 50 basepairs (bp),
preferably at least about 100 bp, more preferably at least about
1000 bp, and usually not more than about 2000 bp of a sequence
homologous with a sequence in the host. In this way, the
probability of legitimate recombination is enhanced, so that the
gene will be integrated into the host and stably maintained by the
host. Desirably, the toxin gene will be in close proximity to the
gene providing for complementation as well as the gene providing
for the competitive advantage. Therefore, in the event that a toxin
gene is lost, the resulting organism will be likely to also lost
the complementing gene and/or the gene providing for the
competitive advantage, so that it will be unable to compete in the
environment with the gene retaining the intact construct.
A large number of transcriptional regulatory regions are available
from a wide variety of microorganism hosts, such as bacteria,
bacteriophage, cyanobacteria, algae, fungi, and the like. Various
transcriptional regulatory regions include the regions associated
with the trp gene, lac gene, gal gene, the .lamda..sub.L and
.lamda..sub.R promoters, the tac promoter, the naturally-occurring
promoters associated with the .delta.-endotoxin gene, where
functional in the host. See for example, U.S. Pat. Nos. 4,332,898;
4,342,832; and 4,356,270 (each of which is specifically
incorporated herein by reference). The termination region may be
the termination region normally associated with the transcriptional
initiation region or a different transcriptional initiation region,
so long as the two regions are compatible and functional in the
host.
Where stable episomal maintenance or integration is desired, a
plasmid will be employed which has a replication system which is
functional in the host. The replication system may be derived from
the chromosome, an episomal element normally present in the host or
a different host, or a replication system from a virus which is
stable in the host. A large number of plasmids are available, such
as pBR322, pACYC184, RSF1010, pR01614, and the like. See for
example, Olson et al. (1982); Bagdasarian et al. (1981), Baum et
al., 1990, and U.S. Pat. Nos. 4,356,270; 4,362,817; 4,371,625, and
5,441,884, each incorporated specifically herein by reference.
The B. thuringiensis gene can be introduced between the
transcriptional and translational initiation region and the
transcriptional and translational termination region, so as to be
under the regulatory control of the initiation region. This
construct will be included in a plasmid, which will include at
least one replication system, but may include more than one, where
one replication system is employed for cloning during the
development of the plasmid and the second replication system is
necessary for functioning in the ultimate host. In addition, one or
more markers may be present, which have been described previously.
Where integration is desired, the plasmid will desirably include a
sequence homologous with the host genome.
The transformants can be isolated in accordance with conventional
ways, usually employing a selection technique, which allows for
selection of the desired organism as against unmodified organisms
or transferring organisms, when present. The transformants then can
be tested for pesticidal activity. If desired, unwanted or
ancillary DNA sequences may be selectively removed from the
recombinant bacterium by employing site-specific recombination
systems, such as those described in U.S. Pat. No. 5,441,884
(specifically incorporated herein by reference).
2.4 Cry3 DNA Segments
A B. thuringiensis cry3* gene encoding a crystal protein having one
or more mutations in one or more regions of the peptide represents
an important aspect of the invention. Preferably, the cry3* gene
encodes an amino acid sequence in which one or more amino acid
residues have been changed based on the methods disclosed herein,
and particularly those changes which have been made for the purpose
of altering the insecticidal activity or specificity of the crystal
protein.
In accordance with the present invention, nucleic acid sequences
include and are not limited to DNA, including and not limited to
cDNA and genomic DNA, genes; RNA, including and not limited to mRNA
and tRNA; antisense sequences, nucleosides, and suitable nucleic
acid sequences such as those set forth in SEQ ID NO:1, SEQ ID NO:3,
SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,
SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID
NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ
ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41,
SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID
NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ
ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69,
SEQ ID NO:99, SEQ ID NO:101, or SEQ ID NO:107, and alterations in
the nucleic acid sequences including alterations, deletions,
mutations, and homologs capable of expressing the B. thuringiensis
modified toxins of the present invention.
As such the present invention also concerns DNA segments, that are
free from total genomic DNA and that encode the novel
synthetically-modified crystal proteins disclosed herein. DNA
segments encoding these peptide species may prove to encode
proteins, polypeptides, subunits, functional domains, and the like
of crystal protein-related or other non-related gene products. In
addition these DNA segments may be synthesized entirely in vitro
using methods that are well-known to those of skill in the art.
As used herein, the term "DNA segment" refers to a DNA molecule
that has been isolated free of total genomic DNA of a particular
species. Therefore, a DNA segment encoding a crystal protein or
peptide refers to a DNA segment that contains crystal protein
coding sequences yet is isolated away from, or purified free from,
total genomic DNA of the species from which the DNA segment is
obtained, which in the instant case is the genome of the
Gram-positive bacterial genus, Bacillus, and in particular, the
species of Bacillus known as B. thuringiensis. Included within the
term "DNA segment", are DNA segments and smaller fragments of such
segments, and also recombinant vectors, including, for example,
plasmids, cosmids, phagemids, phage, viruses, and the like.
Similarly, a DNA segment comprising an isolated or purified crystal
protein-encoding gene refers to a DNA segment which may include in
addition to peptide encoding sequences, certain other elements such
as, regulatory sequences, isolated substantially away from other
naturally occurring genes or protein-encoding sequences. In this
respect, the term "gene" is used for simplicity to refer to a
functional protein-, polypeptide- or peptide-encoding unit. As will
be understood by those in the art, this functional term includes
both genomic sequences, operon sequences and smaller engineered
gene segments that express, or may be adapted to express, proteins,
polypeptides or peptides.
"Isolated substantially away from other coding sequences" means
that the gene of interest, in this case, a gene encoding a
bacterial crystal protein, forms the significant part of the coding
region of the DNA segment, and that the DNA segment does not
contain large portions of naturally-occurring coding DNA, such as
large chromosomal fragments or other functional genes or operon
coding regions. Of course, this refers to the DNA segment as
originally isolated, and does not exclude genes, recombinant genes,
synthetic linkers, or coding regions later added to the segment by
the hand of man.
Particularly preferred DNA sequences are those encoding Cry3Bb.60,
Cry3Bb.11221, Cry3Bb.11222, Cry3Bb.11223, Cry3Bb.11224,
Cry3Bb.11225, Cry3Bb.11226, Cry3Bb.11227, Cry3Bb.11228,
Cry3Bb.11229, Cry3Bb.11230, Cry3Bb.11231, Cry3Bb.11232,
Cry3Bb.11233, Cry3Bb.11234, Cry3Bb.11235, Cry3Bb.11236,
Cry3Bb.11237, Cry3Bb.11238, Cry3Bb.11239, Cry3Bb.11241,
Cry3Bb.11242, Cry3Bb.11032, Cry3Bb.11035, Cry3Bb.11036,
Cry3Bb.11046, Cry3Bb.11048, Cry3Bb.11051, Cry3Bb.11057,
Cry3Bb.11058, Cry3Bb.11081, Cry3Bb.11082, Cry3Bb.11083,
Cry3Bb.11084, Cry3Bb.11095 and Cry3Bb.11098 crystal proteins, and
in particular cry3Bb* genes such as cry3Bb.60, cry3Bb.11221,
cry3Bb.11222, cry3Bb.11223, cry3Bb.11224, cry3Bb.11225,
cry3Bb.11226, cry3Bb.11227, cry3Bb.11228, cry3Bb.11229,
cry3Bb.11230, cry3Bb.11231, cry3Bb.11232, cry3Bb.11233,
cry3Bb.11234, cry3Bb.11235, cry3Bb.11236, cry3Bb.11237,
cry3Bb.11238, cry3Bb.11239, cry3Bb.11241, cry3Bb.11242,
cry3Bb.11032, cry3Bb.11035, cry3Bb.11036, cry3Bb.11046,
cry3Bb.11048, cry3Bb.11051, cry3Bb.11057, cry3Bb.11058,
cry3Bb.11081, cry3Bb.11082, cry3Bb.11083, cry3Bb.11084,
cry3Bb.11095 and cry3Bb.11098. In particular embodiments, the
invention concerns isolated DNA segments and recombinant vectors
incorporating DNA sequences that encode a Cry peptide species that
includes within its amino acid sequence an amino acid sequence
essentially as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ
ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34,
SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID
NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ
ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,
SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID
NO:100, SEQ ID NO:102, or SEQ ID NO:108.
The term "a sequence essentially as set forth in SEQ ID NO:2, SEQ
ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ
ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,
SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID
NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ
ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50,
SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID
NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ
ID NO:70, SEQ ID NO:100, SEQ ID NO:102, or SEQ ID NO:108" means
that the sequence substantially corresponds to a portion of the
sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ
ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,
SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID
NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ
ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46,
SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID
NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ
ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ ID NO:102,
or SEQ ID NO:108, and has relatively few amino acids that are not
identical to, or a biologically functional equivalent of, the amino
acids of any of these sequences. The term "biologically functional
equivalent" is well understood in the art and is further defined in
detail herein (e.g., see Illustrative Embodiments).
Accordingly, sequences that have between about 70% and about 75% or
between about 75% and about 80%, or more preferably between about
81% and about 90%, or even more preferably between about 91% or 92%
or 93% and about 97% or 98% or 99% amino acid sequence identity or
functional equivalence to the amino acids of SEQ ID NO:2, SEQ ID
NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ
ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,
SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID
NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ
ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60,
SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID
NO:70, SEQ ID NO:100, SEQ ID NO:102 or SEQ ID NO:108 will be
sequences that are "essentially as set forth in SEQ ID NO:2, SEQ ID
NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ
ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,
SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID
NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ
ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60,
SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID
NO:70, SEQ ID NO:100, SEQ ID NO:102, or SEQ ID NO:108."
It will be understood that amino acid and nucleic acid sequences
may include additional residues, such as additional N- or
C-terminal amino acids or 5' or 3' sequences, and yet still be
essentially as set forth in one of the sequences disclosed herein,
so long as the sequence meets the criteria set forth above,
including the maintenance of biological protein activity where
protein expression is concerned. The addition of terminal sequences
particularly applies to nucleic acid sequences that may, for
example, include various non-coding sequences flanking either of
the 5' or 3' portions of the coding region or may include various
internal sequences, i.e., introns, which are known to occur within
genes.
The nucleic acid segments of the present invention, regardless of
the length of the coding sequence itself, may be combined with
other DNA sequences, such as promoters, polyadenylation signals,
additional restriction enzyme sites, multiple cloning sites, other
coding segments, and the like, such that their overall length may
vary considerably. It is therefore contemplated that a nucleic acid
fragment of almost any length may be employed, with the total
length preferably being limited by the ease of preparation and use
in the intended recombinant DNA protocol.
For example, nucleic acid fragments may be prepared that include a
short contiguous stretch encoding the peptide sequence disclosed in
SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,
SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID
NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ
ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38,
SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID
NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ
ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66,
SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ ID NO:102, or SEQ ID
NO:108, or that are identical to or complementary to DNA sequences
which encode the peptide disclosed in SEQ ID NO:2, SEQ ID NO:4, SEQ
ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ
ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24,
SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID
NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ
ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52,
SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID
NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ
ID NO:100, SEQ ID NO:102, or SEQ ID NO:108, and particularly the
DNA segments disclosed in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15,
SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID
NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ
ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43,
SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID
NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ
ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99,
SEQ ID NO:101, or SEQ ID NO:107.
Highly preferred nucleic acid segments of the present invention
comprise one or more cry genes of the invention, or a portion of
one or more cry genes of the invention. For certain application,
relatively small contiguous nucleic acid sequences are preferable,
such as those which are about 14 or 15 or 16 or 17 or 18 or 19, or
20, or 30-50, 51-80, 81-100 or so nucleotides in length.
Alternatively, in some embodiments, and particularly those
involving preparation of recombinant vectors, transformation of
suitable host cells, and preparation of transgenic plant cell,
longer nucleic acid segments are preferred, particularly those that
include the entire coding region of one or more cry genes: As such,
the preferred segments may include those that are up to about
20,000 or so nucleotides in length, or alternatively, shorter
sequences such as those about 19,000, about 18,000, about 17,000,
about 16,000, about 15,000, about 14,000, about 13,000, about
12,000, 11,000, about 10,000, about 9,000, about 8,000, about
7,000, about 6,000, about 5,000, about 4,500, about 4,000, about
3,500, about 3,000, about 2,500, about 2,000, about 1,500, about
1,000, about 500, or about 200 or so base pairs in length. Of
course, these numbers are not intended to be exclusionary of all
possible intermediate lengths in the range of from about 20,000 to
about 15 nucleotides, as all of these intermediate lengths are also
contemplated to be useful, and fall within the scope of the present
invention. It will be readily understood that "intermediate
lengths", in these contexts, means any length between the quoted
ranges, such as 14, 15, 16, 17, 18, 19, 20, etc.; 21, 22, 23,
24,25, 26, 27, 28, 29, etc.; 30, 31, 32, 33, 34, 35, 36 . . . etc.;
40, 41, 42, 43, 44 . . . etc., 50, 51, 52,53 . . . etc.; 60, 61,
62, 63 . . . etc., 70, 80, 90, 100, 110, 120, 130 . . . etc.; 200,
210, 220, 230, 240, 250 . . . etc.; including all integers in the
entire range from about 14 to about 10,000, including those
integers in the ranges 200-500; 500-1,000; 1,000-2,000;
2,000-3,000; 3,000-5,000 and the like.
In a preferred embodiment, the nucleic acid segments comprise a
sequence of from about 1800 to about 18,000 base pair in length,
and comprise one or more genes which encode a modified Cry3*
polypeptide disclosed herein which has increased activity against
Coleopteran insect pests.
It will also be understood that this invention is not limited to
the particular nucleic acid sequences which encode peptides of the
present invention, or which encode the amino acid sequence of SEQ
ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ
ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20,
SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID
NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ
ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48,
SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID
NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ
ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ ID NO:102, or SEQ ID
NO:108, including the DNA sequences which are particularly
disclosed in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ
ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35,
SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID
NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ
ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63,
SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ ID
NO:101, or SEQ ID NO:107. Recombinant vectors and isolated DNA
segments may therefore variously include the peptide-coding regions
themselves, coding regions bearing selected alterations or
modifications in the basic coding region, or they may encode larger
polypeptides that nevertheless include these peptide-coding regions
or may encode biologically functional equivalent proteins or
peptides that have variant amino acids sequences.
The DNA segments of the present invention encompass
biologically-functional, equivalent peptides. Such sequences may
arise as a consequence of codon redundancy and functional
equivalency that are known to occur naturally within nucleic acid
sequences and the proteins thus encoded. Alternatively,
functionally-equivalent proteins or peptides may be created via the
application of recombinant DNA technology, in which changes in the
protein structure may be engineered, based on considerations of the
properties of the amino acids being exchanged. Changes designed by
man may be introduced through the application of site-directed
mutagenesis techniques, e.g., to introduce improvements to the
antigenicity of the protein or to test mutants in order to examine
activity at the molecular level
If desired, one may also prepare fusion proteins and peptides,
e.g., where the peptide-coding regions are aligned within the same
expression unit with other proteins or peptides having desired
functions, such as for purification or immunodetection purposes
(e.g., proteins that may be purified by affinity chromatography and
enzyme label coding regions, respectively).
Recombinant vectors from further aspects of the present invention.
Particularly useful vectors are contemplated to be those vectors in
which the coding portion of the DNA segment, whether encoding a
full length protein or smaller peptide, is positioned under the
control of a promoter. The promoter may be in the form of the
promoter that is naturally associated with a gene encoding peptides
of the present invention, as may be obtained by isolating the 5'
non-coding sequences located upstream of the coding segment or
exon, for example, using recombinant cloning and/or PCR.TM.
technology, in connection with the compositions disclosed
herein.
2.5 Vectors, Host Cells, and Protein Expression
In other embodiments, it is contemplated that certain advantages
will be gained by positioning the coding DNA segment under the
control of a recombinant, or heterologous, promoter. As used
herein, a recombinant or heterologous promoter is intended to refer
to a promoter that is not normally associated with a DNA segment
encoding a crystal protein or peptide in its natural environment.
Such promoters may include promoters normally associated with other
genes, and/or promoters isolated from any bacterial, viral,
eukaryotic, or plant cell. Naturally, it will be important to
employ a promoter that effectively directs the expression of the
DNA segment in the cell type, organism, or even animal, chosen for
expression. The use of promoter and cell type combinations for
protein expression is generally known to those of skill in the art
of molecular biology, for example, see Sambrook et al., 1989. The
promoters employed may be constitutive, or inducible, and can be
used under the appropriate conditions to direct high level
expression of the introduced DNA segment, such as is advantageous
in the large-scale production of recombinant proteins or peptides.
Appropriate promoter systems contemplated for use in high-level
expression include, but are not limited to, the Pichia expression
vector system (Pharmacia LKB Biotechnology).
In connection with expression embodiments to prepare recombinant
proteins and peptides, it is contemplated that longer DNA segments
will most often by used, with DNA segments encoding the entire
peptide sequence being most preferred. However, it will be
appreciated that the use of shorter DNA segments to direct the
expression of crystal peptides or epitopic core regions, such as
may be used to generate anti-crystal protein antibodies, also falls
within the scope of the invention. DNA segments that encode peptide
antigens from about 8, 9, 10, or 11 or so amino acids, and up to
and including those of about 30, 40, or 50 or so amino acids in
length, or more preferably, from about 8 to about 30 amino acids in
length, or even more preferably, from about 8 to about 20 amino
acids in length are contemplated to be particularly useful. Such
peptide epitopes may be amino acid sequences which comprise
contiguous amino acid sequence from SEQ ID NO:2, SEQ ID NO:4, SEQ
ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ
ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24,
SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID
NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ
ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52,
SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID
NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ
ID NO:100, SEQ ID NO:102, or SEQ ID NO:108.
2.6 Transformed Host Cells and Transgenic Plants
In one embodiment, the invention provides a transgenic plant having
incorporated into its genome a transgene that encodes a contiguous
amino acid sequence selected from the group consisting of SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6. SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:14. SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ
ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30,
SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID
NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ
ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58,
SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID
NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ ID NO:102, and SEQ ID
NO:108.
A further aspect of the invention is a transgenic plant having
incorporated into its genome a cry3Bb* transgene, provided the
transgene comprises a nucleic acid sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5. SEQ ID NO:7,
SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13. SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ
ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35,
SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID
NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ
ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63,
SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ ID
NO:101, and SEQ ID NO:107. Also disclosed and claimed are progeny
of such a transgenic plant, as well as its seed, progeny from such
seeds, and seeds arising from the second and subsequent generation
plants derived from such a transgenic plant.
The invention also discloses and claims host cells, both native,
and genetically engineered, which express the novel cry3Bb* genes
to produce Cry3Bb* polypeptides. Preferred examples of bacterial
host cells include B. thuringiensis EG11221, EG11222, EG11223,
EG11224, EG11225, EG11226, EG11227, EG11228, EG11229, EG11230,
EG11231, EG11232, EG11233, EG11234, EG11235, EG11236, EG11237,
EG11238, EG11239, EG11241, EG11242, EG11032, EG11035, EG11036,
EG11046, EG11048, EG11051, EG11057, EG11058, EG11081, EG11082,
EG11083, EG11084, EG11095, and EG11098.
Methods of using such cells to produce Cry3* crystal proteins are
also disclosed. Such methods generally involve culturing the host
cell (such as B. thuringiensis EG11221, EG11222, EG11223, EG11224,
EG11225, EG11226, EG11227, EG11228, EG11229, EG11230, EG11231,
EG11232, EG11233, EG11234, EG11235, EG11236, EG11237, EG11238,
EG11239, EG11241, EG11242, EG11032, EG11035, EG11036, EG11046,
EG11048, EG11051, EG11057, EG11058, EG11081, EG11082, EG11083,
EG11084, or EG11095, or EG 11098) under conditions effective to
produce a Cry3* crystal protein, and obtaining the Cry3* crystal
protein from said cell.
In yet another aspect, the present invention provides methods for
producing a transgenic plant which expresses a nucleic acid segment
encoding the novel recombinant crystal proteins of the present
invention. The process of producing transgenic plants is well-known
in the art. In general, the method comprises transforming a
suitable host cell with one or more DNA segments which contain one
or more promoters operatively linked to a coding region that
encodes one or more of the disclosed B. thuringiensis crystal
proteins. Such a coding region is generally operatively linked to a
transcription-terminating region, whereby the promoter is capable
of driving the transcription of the coding region in the cell, and
hence providing the cell the ability to produce the recombinant
protein in vivo. Alternatively, in instances where it is desirable
to control, regulate, or decrease the amount of a particular
recombinant crystal protein expressed in a particular transgenic
cell, the invention also provides for the expression of crystal
protein antisense mRNA. The use of antisense mRNA as a means of
controlling or decreasing the amount of a given protein of interest
in a cell is well-known in the art.
Another aspect of the invention comprises a transgenic plant which
express a gene or gene segment encoding one or more of the novel
polypeptide compositions disclosed herein. As used herein, the term
"transgenic plant" is intended to refer to a plant that has
incorporated DNA sequences, including but not limited to genes
which are perhaps not normally present, DNA sequences not normally
transcribed into RNA or translated into a protein ("expressed"), or
any other genes or DNA sequences which one desires to introduce
into the non-transformed plant, such as genes which may normally be
present in the non-transformed plant but which one desires to
either genetically engineer or to have altered expression.
It is contemplated that in some instances the genome of a
transgenic plant of the present invention will have been augmented
through the stable introduction of one or more Cry3Bb*-encoding
transgenes, either native, synthetically modified, or mutated. In
some instances, more than one transgene will be incorporated into
the genome of the transformed host plant cell. Such is the case
when more than one crystal protein-encoding DNA segment is
incorporated into the genome of such a plant. In certain
situations, it may be desirable to have one, two, three, four, or
even more B. thuringiensis crystal proteins (either native or
recombinantly-engineered) incorporated and stably expressed in the
transformed transgenic plant.
A preferred gene which may be introduced includes, for example, a
crystal protein-encoding a DNA sequence from bacterial origin, and
particularly one or more of those described herein which are
obtained from Bacillus spp. Highly preferred nucleic acid sequences
are those obtained from B. thuringiensis, or any of those sequences
which have been genetically engineered to decrease or increase the
insecticidal activity of the crystal protein in such a transformed
host cell.
Means for transforming a plant cell and the preparation of a
transgenic cell line are well-known in the art, and are discussed
herein. Vectors, plasmids, cosmids, YACs (yeast artificial
chromosomes) and DNA segments for use in transforming such cells
will, of course, generally comprise either the operons, genes, or
gene-derived sequences of the present invention, either native, or
synthetically-derived, and particularly those encoding the
disclosed crystal proteins. These DNA constructs can further
include structures such as promoters, enhancers, polylinkers, or
even gene sequences which have positiely- or negatively-regulating
activity upon the particular genes of interest as desired. The DNA
segment or gene may encode either a native or modified crystal
protein, which will be expressed in the resultant recombinant
cells, and/or which will impart an improved phenotype to the
regenerated plant
Such transgenic plants may be desirable for increasing the
insecticidal resistance of a monocotyledonous or dicotyledonous
plant, by incorporating into such a plant, a transgenic DNA segment
encoding a Cry3Bb* crystal protein which is toxic to coleopteran
insects. Particularly preferred plants include grains such as corn,
wheat, rye, rice, barley, and oats; legumes such as soybeans;
tubers such as potatoes; fiber crops such as flax and cotton; turf
and pasture grasses; ornamental plants; shrubs; trees; vegetables,
berries, citrus, fruits, cacti, succulents, and other
commercially-important crops including garden and houseplants.
In a related aspect, the present invention also encompasses a seed
produced by the transformed plant, a progeny from such seed, and a
seed produced by the progeny of the original transgenic plant,
produced in accordance with the above process. Such progeny and
seeds will have one or more crystal protein transgene(s) stably
incorporated into its genome, and such progeny plants will inherit
the traits afforded by the introduction of a stable transgene in
Mendelian fashion. All such transgenic plants having incorporated
into their genome transgenic DNA segments encoding one or more
Cry3Bb* crystal proteins or polypeptides are aspects of this
invention. Particularly preferred transgenes for the practice of
the invention include nucleic acid segments comprising one or more
cry3Bb* gene(s).
2.7 Biological Functional Equivalents
Modification and changes may be made in the structure of the
peptides of the present invention and DNA segments which encode
them and still obtain a functional molecule that encodes a protein
or peptide with desirable characteristics. The following is a
discussion based upon changing the amino acids of a protein to
create an equivalent, or even an improved, second-generation
molecule. In particular embodiments of the invention, mutated
crystal proteins are contemplated to be useful for increasing the
insecticidal activity of the protein, and consequently increasing
the insecticidal activity and/or expression of the recombinant
transgene in a plant cell. The amino acid changes may be achieved
by changing the codons of the DNA sequence, according to the codons
given in Table 4.
TABLE-US-00004 TABLE 4 Amino Acids Codons Alanine Ala A GCA GCC GCG
GGU Cysteine Cys C UGC UQU Aspartic Acid Asp D GAC GAU Glutamic
Acid Glu B GAA GAG Phenylalanine Phe P UUC UUU Glycine Gly G GGA
GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU
Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CDC CUG CUU
Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC
CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG
CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC
ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine
Tyr Y UAC UAU
For example, certain amino acids may be substituted for other amino
acids in a protein structure without appreciable loss of
interactive binding capacity with structures such as, for example,
antigen-binding regions of antibodies or binding sites on substrate
molecules. Since it is the interactive capacity and nature of a
protein that defines that protein's biological functional activity,
certain amino acid sequence substitutions can be made in a protein
sequence, and, of course, its underlying DNA coding sequence, and
nevertheless obtain a protein with like properties. It is thus
contemplated by the inventors that various changes may be made in
the peptide sequences of the disclosed compositions, or
corresponding DNA sequences which encode said peptides without
appreciable loss of their biological utility or activity.
In making such changes, the hydropathic index of amino acids may be
considered. The importance of the hydropathic amino acid index in
conferring interactive biologic function on a protein is generally
understood in the art (Kyte and Doolittle, 1982, incorporate herein
by reference). It is accepted that the relative hydropathic
character of the amino acid contributes to the secondary structure
of the resultant protein, which in turn defines the interaction of
the protein with other molecules, for example, enzymes, substrates,
receptors, DNA, antibodies, antigens, and the like.
Each amino acid has been assigned a hydropathic index on the basis
of their hydrophobicity and charge characteristics (Kyte and
Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
It is known in the art that certain amino acids may be substituted
by other amino acids having a similar hydropathic index or score
and still result in a protein with similar biological activity,
i.e., still obtain a biological functionally equivalent protein. In
making such changes, the substitution of amino acids whose
hydropathic indices are within .+-.2 is preferred, those which are
within .+-.1 are particularly preferred, and those within .+-.0.5
are even more particularly preferred.
It is also understood in the art that the substitution of like
amino acids can be made effectively on the basis of hydrophilicity.
U.S. Pat. No. 4,554,101, specifically incorporated herein by
reference, states that the greatest local average hydrophilicity of
a protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with a biological property of the protein.
As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4).
It is understood that an amino acid can be substituted for another
having a similar hydrophilicity value and still obtain a
biologically equivalent, and in particular, an immunologically
equivalent protein. In such changes, the substitution of amino
acids whose hydrophilicity values are within .+-.2 is preferred,
those which are within .+-.1 are particularly preferred, and those
within .+-.0.5 are even more particularly preferred.
As outlined above, amino acid substitutions are generally therefore
based on the relative similarity of the amino acid side-chain
substituents, for example, their hydrophobicity, hydrophilicity,
charge, size, and the like. Exemplary substitutions which take
various of the foregoing characteristics into consideration are
well known to those of skill in the art and include: arginine and
lysine; glutamate and aspartate; serine and threonine; glutamine
and asparagine; and valine, leucine and isoleucine.
3.0 BRIEF DESCRIPTION OF THE DRAWINGS
The drawings form part of the present specification and are
included to further demonstrate certain aspects of the present
invention. The invention may be better understood by reference to
one or more of these drawings in combination with the detailed
description of specific embodiments presented here.
FIG. 1. Schematic representation of the monomeric structure of
Cry3Bb.
FIG. 2. Stereoscopic view of the monomeric structure of Cry3Bb with
associated water molecules (represented by dots).
FIG. 3A. Schematic representation of domain 1 of Cry3Bb
FIG. 3B. Diagram of the positions of the 7 helices that comprise
domain 1.
FIG. 4. Domain 1 of Cry3Bb is organized into seven .alpha. helices
illustrated in FIG. 3A (schematic representation) and FIG. 3B
(schematic diagram). The .alpha. helices and amino acids residues
are shown.
FIG. 5A. Schematic representation of domain 2 and Cry3Bb.
FIG. 5B. Diagram of the positions of the 11 .beta. strands that
compose the 3 .beta.sheets of domain 2.
FIG. 6. Domain 2 of Cry3Bb is a collection of three anti-parallel
.beta. sheets illustrated in FIG. 5. The amino acids that define
these sheets is listed below (.alpha.8, amino aids 322-328, also is
included in domain 2):
FIG. 7A. Schematic representation of domain 3 of Cry3Bb.
FIG. 7B. Diagram of the positions of the .beta. strands that
comprise domain 3.
FIG. 8. Domain 3 (FIG. 7) is a loosely organized collection of
.beta. strands and loops; no .beta. sheets are present. The .beta.
strands contain the amino acids limited below:
FIG. 9A. A "side" view of the dimeric structure of Cry3Bb. The
helical bundles of domains 1 can be seem in the middle of the
molecule.
FIG. 9B. A "top" view of the dimeric structure of Cry3Bb. The
helical bundles of domains 1 can be seem in the middle of the
molecule.
FIG. 10. A graphic representation of the growth in conductance with
time of channels formed by Cry3A and Cry3Bb in planar lipid
bilayers. Cry3A forms channels with higher conductances much more
rapidly than Cry3Bb.
FIG. 11. A map of pEG1701 which contains the Cry3Bb gene with the
cry1F terminator.
FIG. 12. The results of replicated 1-dose assays against SCRW
larvae of Cry3Bb proteins altered in the 1B2,3 region.
FIG. 13. The results of replicated, 1-dose assays against SCRW
larvae of Cry3Bb proteins altered in the 1B6, 7 region.
FIG. 14. The results of replicated, 1-dose screens against SCRW
larvae of Cry3Bb proteins altered in the 1B10,11 region.
FIG. 15. Single channel recordings of channels formed by
Cry3Bb.11230 and WT Cry3Bb in planar lipid bilayers. Cry3Bb. 11230
forms channels with well resolved open and closed states while
Cry3Bb rarely does.
FIG. 16. Single channel recordings of channels formed by Cry3Bb and
Cry3Bb.60, a truzncated form of Cry3Bb. Cry3Bb.60 forms channels
more quickly than Cry3Bb and, unlike Cry3Bb, produces channels with
well resolved open and closed states.
FIG. 17A. Sequence alignment of the amino acid sequence of Cry3A,
Cry3B, and Cry3C.
FIG. 17B. Shown is a continuation of alignment of the amino acid
sequence of Cry3A, Cry3B, and Cry3C shown in FIG. 17A.
FIG. 17C. Shown is a continuation of alignment of the amino acid
sequence of Cryp3A, Cry3B, and Cry3C shown in FIG. 17A.
4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The invention defines new B. thuringiensis (Bt) insecticidal
.delta.-endotoxin proteins and the biochemical and biophysical
strategies used to design the new proteins. Delta-endotoxins are a
class of insecticidal proteins produced by B. thuringiensis that
form cation-selective channels in planar lipid bilayers (English
and Slatin, 1992). The new .delta.-endotoxins are based on the
parent structure of the coleopteran-active, .delta.-endotoxin
Cry3Bb. Like other members of the coleopteran-active class of
.delta.-endotoxins, including Cry3A and Cry3B, Cry3Bb exhibits
excellent insecticidal activity against the Colorado Potato Beetle
(Leptinotarsa decemlineata). However, unlike Cry3A and Cry3B,
Cry3Bb is also active against the southern corn rootworm or SCRW
(Diabrotica undecimpunctata howardi Barber) and the western corn
rootworm or WCRW (Diabrotica virgifera virfigera LeConte). The new
insecticidal proteins described herein were specifically designed
to improve the biological activity of the parent Cry3Bb protein. In
addition, the design strategies themselves are novel inventions
capable of being applied to and improving B. thuringiensis
.delta.-endotoxins in general. B. thuringiensis .delta.-endotoxins
are also members of a larger class of bacterial toxins that form
ion channels (see English and Slatin 1992, for a review). The
inventors, therefore, believe that these design strategies can also
be applied to any biologically active, channel-forming protein to
improve its biological properties.
The designed Cry3Bb proteins were engineered using one or more of
the following strategies including (1) identification and
alteration of protease-sensitive sites and proteolytic processing;
(2) analysis and manipulation of bound water; (3) manipulation of
hydrogen bonds around mobile regions; (4) loop analysis and loop
redesign around flexible helices; (5) loop design around .beta.
strands and .beta. sheets; (6) identification and redesign of
complex electrostatic surfaces; (7) identification and removal of
metal binding sites; (8) alteration of quatenary structure; (9)
identification and design of structural residues; and (10)
combinations of any and all sites defined by stragegies 1-9. These
design strategies permit the identification and redesign of
specific sites on Cry3Bb, ultimately creating new proteins with
improved insecticidal activities. These new proteins are designated
Cry3Bb designed proteins and are named Cry3Bb followed by a period
and a suffix (e.g., Cry3Bb.60, Cry3Bb.11231). The new proteins are
listed in Table 2 along with the specific sites on the molecule
that were modified, the amino-acid sequence changes at those sites
that improve biological activity, the improved insecticidal
activities and the design method used to identify that specific
site.
4.1 Some Advantages of the Invention
Mutagenesis studies with cry genes have failed to identify a
significant number of mutant crystal proteins which have improved
broad-spectrum insecticidal activity, that is, with improved
toxicity towards a range of insect pest species. Since agricultural
crops are typically threatened by more than one insect pest species
at any given time, desirable mutant crystal proteins are preferably
those that exhibit improvements in toxicity towards multiple insect
pest species. Previous failures to identify such mutants may be
attributed to the choice of sites targeted for mutagenesis. For
example, with respect to the related protein, Cry1C, sites within
domain 2 and domain 3 have been the principal targets of
mutagenesis efforts, primarily because these domains are believed
to be important for receptor binding and in determining
insecticidal specificity (Aronson et al., 1995; Chen et al. 1993;
de Maagd et al., 1996; Lee et al., 1992; Lee et al., 1995; Lu et
al., 1994; Smedley and Ellar, 1996; Smith and Ellar, 1994;
Rajamohan et al., 1995; Rajamohan et al., 1996)
In contrast, the present inventors reasoned that the toxicity of
Cry3 proteins, and specifically the toxicity of the Cry3Bb protein,
may be improved against a broader array of target pests by
targeting regions involved in ion channel function rather than
regions of the molecule directly involved in receptor interactions,
namely domains 2 and 3. Accordingly, the inventors opted to target
regions within domain 1 of Cry3Bb for mutagenesis for the purpose
of isolating Cry3Bb mutants with improved broad spectrum toxicity.
Indeed, in the present invention, Cry3Bb mutants are described that
show improved toxicity towards several coleopteran pests.
At least one, and probably more than one, .alpha. helix of domain 1
is involved in the formation of ion channels and pores within the
insect midgut epithelium (Gazit and Shai, 1993; Gazit and Shai,
1995). Rather than target for mutagenesis the sequences encoding
the .alpha. helices of domain 1 as others have (Wu and Aronson,
1992; Aronson et al., 1995; Chen et al., 1995), the present
inventors opted to target exclusively sequences encoding amino acid
residues adjacent to or lying within the predicted loop regions of
Cry3Bb that separate these .alpha. helices. Amino acid residues
within these loop regions or amino acid residues capping the end of
an .alpha. helix and lying adjacent to these loop regions may
affect the spatial relationships among these .alpha. helices.
Consequently, the substitution of these amino acid residues may
result in subtle changes in tertiary structure, or even quaternary
structure, that positively impact the function of the ion channel.
Amino acid residues in the loop regions of domain 1 are exposed to
the solvent and thus are available for various molecular
interactions. Altering these amino acids could result in greater
stability of the protein by eliminating or occluding
protease-sensitive sites. Amino acid substitutions that change the
surface charge of domain 1 could alter ion channel efficiency or
alter interactions with the brush border membrane or with other
portions of the toxin molecule, allowing binding or insertion to be
more effective.
According to this invention, base substitutions are made in the
underlying cry3Bb nucleic acid residues in order to change
particular codons of the corresponding polypeptides, and
particularly, in those loop regions between .alpha.-helices. The
insecticidal activity of a crystal protein ultimately dictates the
level of crystal protein required for effective insect control. The
potency of an insecticidal protein should be maximized as much as
possible in order to provide for its economic and efficient
utilization in the field. The increased potency of an insecticidal
protein in a bioinsecticide formulation would be expected to
improve the field performance of the bioinsecticide product.
Alternatively, increased potency of an insecticidal protein in a
bioinsecticide formulation may promote use of reduced amounts of
bioinsecticide per unit area of treated crop, thereby allowing for
more cost-effective use of the bioinsecticide product. When
expressed in planta, the production of crystal proteins with
improved insecticidal activity can be expected to improve plant
resistance to susceptible insect pests.
4.2 Methods for Culturing B. Thuringiensis to Produce Crystal
Proteins
The B. thuringiensis strains described herein may be cultured using
standard known media and fermentation techniques. Upon completion
of the fermentation cycle, the bacteria may be harvested by first
separating the B. thuringiensis spores and crystals from the
fermentation broth by means well known in the art. The recovered B.
thuringiensis spores and crystals can be fonnulated into a wettable
powder, a liquid concentrate, granules or other formulations by the
addition of surfactants, dispersants, inert carriers and other
components to facilitate handling and application for particular
target pests. The formulation and application procedures are all
well known in the art.
4.3 Recombinate Host Cells for Expression of Cry* Genes
The nucleotide sequences of the subject invention can be introduced
into a wide variety of microbial hosts. Expression of the toxin
gene results, directly or indirectly, in the intracellular
production and maintenance of the pesticide. With suitable hosts,
e.g., Pseudomonas, the microbes can be applied to the sites of
coleopteran insects where they will proliferate and be ingested by
the insects. The result is a control of the unwanted insects.
Alternatively, the microbe hosting the toxin gene can be treated
under conditions that prolong the activity of the toxin produced in
the cell. The treated cell then can be applied to the environment
of target pest(s). The resulting product retains the toxicity of
the B. thuringiensis toxin.
Suitable host cells, where the pesticide-containing cells will be
treated to prolong the activity of the toxin in the cell when the
then treated cell is applied to the environment of target pest(s),
may include either prokaryotes or eukaryotes, normally being
limited to those cells which do not produce substances toxic to
higher organisms, such as mammals. However, organisms which produce
substances toxic to higher organisms could be used, where the toxin
is unstable or the level of application sufficiently low as to
avoid any possibility or toxicity to a mammalian host. As hosts, of
particular interest will be the prokaryotes and the lower
eukaryotes, such as fungi. Illustrative prokaryotes, both
Gram-negative and Gram-positive, include Enterobacteriaceae, such
as Escherichia, Erwinia, Shigella, Salmonella, and Proteus;
Bacillaceae; Rhizobiceae, such as Rhozibium; Spirillaceae, such as
photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,
Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such
as Pseudomonas and Acetobacter; Azotobacteraceae, Actinomycetales,
and Nitrobacteraceae. Among eukaryotes are fungi, such as
Phycomycetes and Ascomycetes, which includes yeast, such as
Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeast,
such as Rhodotorula, Aureobasidium, Sporobolomyces, and the
like.
Characteristics of particular interest in selecting a host cell for
purposes of production include ease of introducing the B.
thuringiensis gene into the host, availability of expression
systems, efficiency of expression, stability of the pesticide in
the host, and the presence of auxiliary genetic capabilities.
Characteristics of interest for use as a pesticide micro-capsule
include protective qualities for the pesticide, such as thick cell
walls, pigmentation, and intracellular packaging or formation of
inclusion bodies; leaf affinity; lack of mammalian toxicity;
attractiveness to pests for ingestion; ease of killing and fixing
without damage to the toxin; and the like. Other considerations
include ease of formulation and handling, economics, storage
stability, and the like.
Host organisms of particular interest include yeast, such as
Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., and
Sporobolomyces sp.; phylloplane organisms such as Pseudomonas sp.,
Erwinia sp. and Flavobacterium sp.; or such other organisms as
Escherichia, Lactobacillus sp., Bacillus sp., Streptomyces sp., and
the like. Specific organisms include Pseudomonas aeruginosa,
Pseudomonas fluorescens, Saccharomyces cerevisiae, B.
thuringiensis, Escherichia coli, B. subtilis, B. megaterium, B.
cereus, Streptomyces lividans and the like.
Treatment of the microbial cell, e.g., a microbe containing the B.
thuringiensis toxin gene, can be by chemical or physical means, or
by a combination of chemical and/or physical means, so long as the
technique does not deleteriously affect the properties of the
toxin, nor diminish the cellular capability in protecting the
toxin. Examples of chemical reagents are halogenating agents,
particularly halogens of atomic no. 17-80. More particularly,
iodine can be used under mild conditions and for sufficient time to
achieve the desired results. Other suitable techniques include
treatment with aldehydes, such as formaldehyde and glutaraldehye;
anti-infectives, such as zephiran chloride and cetylpyridinium
chloride; alcohols, such as isopropyl and ethanol; various
histologic fixatives, such as Lugol's iodine, Bouin's fixative, and
Helly's fixatives, (see e.g., Humason, 1967); or a combination of
physical (heat) and chemical agents that preserve and prolong the
activity of the toxin produced in the cell when the cell is
administered to the host animal. Examples of physical means are
short wavelength radiation such as .gamma.-radiation and
X-radiation, freezing, UV irradiation, lyophilization, and the
like. The cells employed will usually be intact and be
substantially in the proliferative form when treated, rather than
in a spore form, although in some instances spores may be
employed.
Where the B. thuringiensis toxin gene is introduced via a suitable
vector into a microbial host, and said host is applied to the
environment in a living state, it is essential that certain host
microbes be used. Microorganism hosts are selected which are known
to occupy the "phytosphere" (phylloplane, phyllosphere,
rhizosphere, and/or rhizoplane) of one or more crops of interest.
These microorganisms are selected so as to be capable of
successfully competing in the particular environment (crop and
other insect habitats) with the wild-type microorganisms, provide
for stable maintenance and expression of the gene expressing the
polypeptide pesticide, and, desirably, provide for improved
protection of the pesticide from environmental degradation and
inactivation.
A large number of microorganisms are known to inhabit the
phylloplane (the surface of the plant leaves) and/or the
rhizosphere (the soil surrounding plant roots) of a wide variety of
important crops. These microorganisms include bacteria, algae, and
fungi. Of particular interest are microorganisms, such as bacteria,
e.g., genera Bacillus (including the species and subspecies B.
thuringiensis kurstaki HD-1, B. thuringiensis kurstaki HD-73, B.
thuringiensis sotto, B. thuringiensis berliner, B. thuringiensis
thuringiensis, B. thuringiensis tolworthi, B. thuringiensis
dendrolimus, B. thuringiensis alesti, B. thuringiensis galleriae,
B. thuringiensis aizawai, B. thuringiensis subtoxicus, B.
thuringiensis entomocidus, B. thuringiensis tenebrionis and B.
thuringiensis san diego); Pseudomonas, Erwinia, Serratia,
Klebsiella, Zanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas,
Methylophilius, Agrobacterium, Acetobacter, Lactobacillus,
Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fingi,
particularly yeast, e.g., genera Saccharomyces, Cryptococcus,
Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of
particular interest are such phytosphere bacterial species as
Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,
Acetobacter xylinum, Agrobacterium tumefaciens, Rhodobacter
sphaeroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes
eutrophus, and Azotobacter vinlandii; and phytosphere yeast species
such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,
Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces
rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S.
odorus, Kluyveromyces veronae, and Aureobasidium pollulans.
4.4 Definitions
In accordance with the present invention, nucleic acid sequences
include and are not limited to DNA (including and not limited to
genomic or extragenomic DNA), genes, RNA (including and not limited
to mRNA and tRNA), nucleosides, and suitable nucleic acid segments
either obtained from native sources, chemically synthesized,
modified, or otherwise prepared by the hand of man. The following
words and phrases have the meanings set forth below.
A, an: In accordance with long standing patent law convention, the
words "a" and "an" when used in this application, including the
claims, denotes "one or more".
Broad-spectrum: Refers to a wide range of insect species.
Broad-spectrum activity: The toxicity towards a wide range of
insect species.
Expression: The combination of intracellular processes, including
transcription and translation undergone by a coding DNA molecule
such as a structural gene to produce a polypeptide.
Insecticidal activity: The toxicity towards insects.
Insecticidal specificity: The toxicity exhibited by a crystal
protein or proteins, microbe or plant, towards multiple insect
species.
Intraorder specificity: The toxicity of a particular crystal
protein towards insect species within an Order of insects (e.g.,
Order Coleoptera).
Interorder specificity: The toxicity of a particular crystal
protein towards insect species of different Orders (e.g., Orders
Coleoptera and Diptera).
LC.sub.50: The lethal concentration of crystal protein that causes
50% mortality of the insects treated.
LC.sub.95: The lethal concentration of crystal protein that causes
95% mortality of the insects treated.
Promoter: A recognition site on a DNA sequence or group of DNA
sequences that provide an expression control element for a
structural gene and to which RNA polymerase specifically binds and
initiates RNA synthesis (transcription) of that gene.
Regeneration: The process of growing a plant from a plant cell
(e.g., plant protoplast or explant).
Structural gene: A gene that is expressed to produce a
polypeptide.
Transformation: A process of introducing an exogenous DNA sequence
(e.g., a vector, a recombinant DNA molecule) into a cell or
protoplast in which that exogenous DNA is incorporated into a
chromosome or is capable of autonomous replication.
Transformed cell: A cell whose DNA has been altered by the
introduction of an exogenous DNA molecule into that cell.
Transgenic cell: Any cell derived or regenerated from a transformed
cell or derived from a transgenic cell. Exemplary transgenic cells
include plant calli derived from a transformed plant cell and
particular cells such as leaf, root, stem, e.g., somatic cells, or
reproductive (germ) cells obtained from a transgenic plant.
Transgenic plant: A plant or progeny thereof derived from a
transformed plant cell or protoplast, wherein the plant DNA
contains an introduced exogenous DNA molecule not originally
present in a native, non-transgenic plant of the same strain. The
terms "transgenic plant" and "transformed plant" have sometimes
been used in the art as synonymous terms to define a plant whose
DNA contains an exogenous DNA molecule. However, it is thought more
scientifically correct to refer to a regenerated plant or callus
obtained from a transformed plant cell or protoplast as being a
transgenic plant, and that usage will be followed herein.
Vector: A DNA molecule capable of replication in a host cell and/or
to which another DNA segment can be operatively linked so as to
bring about replication of the attached segment. A plasmid is an
exemplary vector.
As used herein, the designations "CryIII" and "Cry3" are
synonymous, as are the designations "CryIIIB2" and "Cry3Bb."
Likewise, the inventors have utilized the generic term Cry3Bb* to
denote any and all Cry3Bb variants which comprise amino acid
sequences modified in the protein. Similarly, cry3Bb* is meant to
denote any and all nucleic acid segments and/or genes which encode
a Cry3Bb* protein, etc.
4.5 Preparation of Cry3* Polynucleotides
Once the structure of the desired peptide to be mutagenized has
been analyzed using one or more of the design strategies disclosed
herein, it will be desirable to introduce one or more mutations
into either the protein or, alternatively, into the DNA sequence
encoding the protein for the purpose of producing a mutated protein
with altered bioinsecticidal properties.
To that end, the present invention encompasses both site-specific
mutagenesis methods and random mutagenesis of a nucleic acid
segment encoding a crystal protein in the manner described herein.
In particular, methods are disclosed for the mutagenesis of nucleic
acid segments encoding the amino acid sequences using one or more
of the design strategies described herein. Using the assay methods
described herein, one may then identify mutants arising from these
procedures which have improved insecticidal properties or altered
specificity, either intraorder or interorder.
The means for mutagenizing a DNA segment encoding a crystal protein
are well-known to those of skill in the art. Modifications may be
made by random, or site-specific mutagenesis procedures. The
nucleic acid may be modified by altering its structure through the
addition or deletion of one or more nucleotides from the
sequence.
Mutagenesis may be performed in accordance with any of the
techniques known in the art such as and not limited to synthesizing
an oligonucleotide having one or more mutations within the sequence
of a particular crystal protein. A "suitable host" is any host
which will express Cry3Bb, such as and not limited to B.
thuringiensis and E. coli. Screening for insecticidal activity, in
the case of Cry3Bb includes and is not limited to coleopteran-toxic
activity which may be screened for by techniques known in the
art.
In particular, site-specific mutagenesis is a technique useful in
the preparation of individual peptides, or biologically functional
equivalent proteins or peptides, through specific mutagenesis of
the underlying DNA. The technique further provides a ready ability
to prepare and test sequence variants, for example, incorporating
one or more of the foregoing considerations, by introducing one or
more nucleotide sequence changes into the DNA. Site-specific
mutagenesis allows the production of mutants through the use of
specific oligonucleotide sequences which encode the DNA sequence of
the desired mutation, as well as a sufficient number of adjacent
nucleotides, to provide a primer sequence of sufficient size and
sequence complexity to form a stable duplex on both sides of the
deletion junction being traversed. Typically, a primer of about 17
to about 75 nucleotides or more in length is preferred, with about
10 to about 25 or more residues on both sides of the junction of
the sequence being altered.
In general, the technique of site-specific mutagenesis is well
known in the art, as exemplified by various publications. As will
be appreciated, the technique typically employs a phage vector
which exists in both a single stranded and double stranded form.
Typical vectors useful in site-directed mutagenesis include vectors
such as the M13 phage. These phage are readily commercially
available and their use is generally well known to those skilled in
the art. Double stranded plasmids are also routinely employed in
site directed mutagenesis which eliminates the step of transferring
the gene of interest from a plasmid to a phage.
In general, site-directed mutagenesis in accordance herewith is
performed by first obtaining a single-stranded vector or melting
apart of two strands of a double stranded vector which includes
within its sequence a DNA sequence which encodes the desired
peptide. An oligonucleotide primer bearing the desired mutated
sequence is prepared, generally synthetically. This primer is then
annealed with the single-stranded vector, and subjected to DNA
polymerizing enzymes such as E. coli polymerase 1 Klenow fragment,
in order to complete the synthesis of the mutation-bearing strand.
Thus, a heteroduplex is formed wherein one strand encodes the
original non-mutated sequence and the second strand bears the
desired mutation. This heteroduplex vector is then used to
transform or transfect appropriate cells, such as E. coli cells,
and clones are selected which include recombinant vectors bearing
the mutated sequence arrangement. A genetic selection scheme was
devised by Kunkel et al (1987) to enrich for clones incorporating
the mutagenic oligonucleotide. Alternatively, the use of PCR.TM.
with commercially available thermostable enzymes such as Taq
polymerase may be used to incorporate a mutagenic oligonucleotide
primer into an amplified DNA fragment that can then be cloned into
an appropriate cloning or expression vector. The PCR.TM.-mediated
mutagenesis procedure of Tomic et al. (1990) and Upender et al.
(1995) provide two examples of such protocols. A PCR.TM. employing
a thermostable ligase in addition to a thernostable polymerase may
also be used to incorporate a phosphorylated mutagenic
oligonucleotide into an amplified DNA fragment that may then be
cloned into an appropriate cloning or expression vector. The
mutagenesis procedure described by Michael (1994) provides an
example of one such protocol.
The preparation of sequence variants of the selected
peptide-encoding DNA segments using site-directed mutagenesis is
provided as a means of producing potentially useful species and is
not meant to be limiting as there are other ways in which sequence
variants of peptides and the DNA sequences encoding them may be
obtained. For example, recombinant vectors encoding the desired
peptide sequence may be treated with mutagenic agents, such as
hydroxylamine, to obtain sequence variants.
As used herein, the term "oligonucleotide directed mutagenesis
procedure" refers to template-dependent processes and
vector-mediated propagation which result in an increase in the
concentration of a specific nucleic acid molecule relative to its
initial concentration, or in an increase in the concentration of a
detectable signal, such as amplification. As used herein, the term
"oligonucleotide directed mutagenesis procedure" is intended to
refer to a process that involves the template-dependent extension
of a primer molecule. The term template dependent process refers to
nucleic acid synthesis of an RNA or a DNA molecule wherein the
sequence of the newly synthesized strand of nucleic acid is
dictated by the well-known rules of complementary base pairing
(see, for example, Watson, 1987). Typically, vector mediated
methodologies involve the introduction of the nucleic acid fragment
into a DNA or RNA vector, the clonal amplification of the vector,
and the recovery of the amplified nucleic acid fragment. Examples
of such methodologies are provided by U.S. Pat. No. 4,237,224,
specifically incorporated herein by reference in its entirety
A number of template dependent processes are available to amplify
the target sequences of interest present in a sample. One of the
best known amplification methods is the polymerase chain reaction
(PCR.TM.) which is descrived in detail in U.S. Pat. Nos. 4,683,195,
4,683,202 and 4,800,159 (each of which is specifically incorporated
herein by reference in its entirety). Briefly, in PCR.TM., two
primer sequences are prepared which are complementary to regions on
opposite complementary strands of the target sequence. An excess of
deoxynucleoside triphosphates are added to a reaction mixture along
with a DNA polymerase (e.g., Taq polymerase). If the target
sequence is present in a sample, the primers will bind to the
target and the polymerase will cause the primers to be extended
along the target sequence by adding on nucleotides. By raising and
lowering the temperature of the reaction mixture, the extended
primers will dissociate from the target to form reaction products,
excess primers will bind to the target and to the reaction products
and the process is repeated. Preferably a reverse transcriptase
PCR.TM. amplification procedure may be performed in order to
quantify the amount of mRNA amplified. Polymerase chain reaction
methodologies are well known in the art.
Another method for amplification is the ligase chain reaction
(referred to as LCR), disclosed in Eur. Pat. Appl. Publ. No.
320,308, incorporated herein by reference in its entirety. In LCR,
two complementary probe pairs are prepared, and in the presence of
the target sequence, each pair will bind to opposite complementary
strands of the target such that they abut. In the presence of a
ligase, the two probe pairs will link to form a single unit. By
temperature cycling, as in PCR.TM., bound ligated units dissociate
from the target and then serve as "target sequences" for ligation
of excess probe pairs. U.S. Pat. No. 4,883,750, specifically
incorporated herein by reference in its entirety, describes an
alternative method of amplification similar to LCR for binding
probe pairs to a taret sequence.
Qbeta Replicase.TM., described in Intl. Pat. Appl. Publ. No.
PCT/US87/00880, incorporated herein by reference in its entirety,
may also be used as still another amplification method in the
present invention. In this method, a replicative sequence of RNA
which has a region complementary to that of a target is added to a
sample in the presence of an RNA polymerase. The polymerase will
copy the replicative sequence which can then be detected.
An isothermal amplification method, in which restriction
endonucleases and ligases are used to achieve the amplification of
target molecules that contain nucleotide 5'-[.alpha.-thio]
triphosphates in one strand of a restriction site (Walker et al.,
1992, incorporated herein by reference in its entirety), may also
be useful in the amplification of nucleic acids in the present
invention.
Strand Displacement Amplification (SDA) is another method of
carrying out iso-thermal amplification of nucleic acids which
involves multiple rounds of strand displacement and synthesis,
i.e., nick translation. A similar method, called Repair Chain
Reaction (RCR) is another method of amplification which may be
useful in the present invention and is involves annealing several
probes throughout a region targeted for amplification, followed by
a repair reaction in which only two of the four bases are present.
The other two bases can be added as biotinylated derivatives for
easy detection. A similar approach is used in SDA
Sequences can also be detected using a cyclic probe reaction (CPR).
In CPR, a probe having 3' and 5' end sequences of non-Cry-specific
DNA and an internal sequence of a Cry-specific RNA is hybridized to
DNA which is present in a sample. Upon hybridization, the reaction
is treated with RNaseH, and the products of the probe identified as
distinctive products generating a signal which are released after
digestion. The original template is annealed to another cycling
probe and the reaction is repeated. Thus, CPR involves amplifying a
signal generated by hybridization of a probe to a cry-specific
expressed nucleic acid
Still other amplification methods described in Great Britain Pat.
Appl. No. 2 202 328, and in Intl. Pat. Appl. Publ. No.
PCTIUS89/01025, each of which is incorporated herein by reference
in its entirety, may be used in accordance with the present
invention. In the former application, "modified" primers are used
in a PCT.TM. like, template and enzyme dependent synthesis. The
primers may be modified by labeling with a capture moiety (e.g.,
biotin) and/or a detector moiety (e.g., enzyme). In the latter
application, an excess of labeled probes are added to a sample. In
the presence of the target sequence, the probe binds and is cleaved
catalytically. After cleavage, the target sequence is released
intact to be bound by excess probe. Cleavage of the labeled probe
signals the presence of the target sequence
Other nucleic acid amplification procedures include
transcription-based amplification systems (TAS) (Kwoh et al., 1989;
Intl. Pat. Appl. Publ. No. WO 88/10315, incorporated herein by
reference in its entirety), including nucleic acid sequence based
amplification (NASBA) and 3SR. In NASBA, the nucleic acids can be
prepared for amplification by standard phenol/chloroform
extraction, heat denaturation of a sample, treatment with lysis
buffer and minispin columns for isolation of DNA and RNA or
guanidinium chloride extraction of RNA. These amplification
techniques involve annealing a primer which has crystal
protein-specific sequences. Following polymerization, DNA/RNA
hybrids are digested with RNase H while double stranded DNA
molecules are heat denatured again. In either case the single
stranded DNA is made fully double stranded by addition of second
crystal protein-specific primer, followed by polymerization. The
double stranded DNA molecules are then multiply transcribed by a
polymerase such as T7 or SP6. In an isothermal cyclic reaction, the
RNAs are reverse transcribed into double stranded DNA, and
transcribed once against with a polymerase such as T7 or SP6. The
resulting products, whether truncated or complete, indicate crystal
protein-specific sequences.
Eur. Pat. Appl. Publ. No. 329,822, incorporated herein by reference
in its entirety, disclose a nucleic acid amplification process
involving cyclically synthesizing single-stranded RNA ("ssRNA"),
ssDNA, and double-stranded DNA (dsDNA), which may be used in
accordance with the present invention. The ssRNA is a first
template for a first primer oligonucleotide, which is elongated by
reverse transcriptase (RNA-dependent DNA polymerase). The RNA is
then removed from resulting DNA:RNA duplex by the action of
ribonuclease H (RNase H, an RNase specific for RNA in a duplex with
either DNA or RNA). The resultant ssDNA is a second template for a
second primer, which also includes the sequences of an RNA
polymerase promoter (exemplified by T7 RNA polymerase) 5' to its
homology to its template. This primer is then extended by DNA
polymerase (exemplified by the large "Klenow" fragment of E. coli
DNA polymerase I), resulting as a double-stranded DNA ("dsDNA")
molecule, having a sequence identical to that of the original RNA
between the primers and having additionally, at one end, a promoter
sequence. This promoter sequence can be used by the appropriate RNA
polymerase to make many RNA copies of the DNA. These copies can
then re-enter the cycle leading to very swift amplification. With
proper choice of enzymes, this amplification can be done
isothermally without addition of enzymes at each cycle. Because of
the cyclical nature of this process, the starting sequence can be
chosen to be in the form of either DNA or RNA
Intl. Pat. Appl. Publ. No. WO 89/06700, incorporated herein by
reference in its entirety, disclose a nucleic acid sequence
amplification scheme based on the hybridization of a
promoter/primer sequence to a target single-stranded DNA ("ssDNA")
followed by transcription of many RNA copies of the sequence. This
scheme is not cyclic; i.e., new templates are not produced from the
resultant RNA transcripts. Other amplification methods include
"RACE" (Frohman, 1990), and "one-sided PCR.TM." (Ohara, 1989) which
are well-known to those of skill in the art.
Methods based on ligation of two (or more) oligonucleotides in the
presence of nucleic acid having the sequence of the resulting
"di-oligonucleotide", thereby amplifying the di-oligonucleotide (Wu
and Dean, 1996, incorporated herein by reference in its entirety),
may also be used in the amplification of DNA sequences of the
present invention.
4.6 Phage-resistant Variants
In certain embodiments, one may desired to prepare one or more
phage resistant variants of the B. thuringiensis mutants prepared
by the methods described herein. To do so, an aliquot of a phage
lysate is spread onto nutrient agar and allowed to dry. An aliquot
of the phage sensitive bacterial strain is then plated directly
over the dried lysate and allowed to dry. The plates are incubated
at 30.degree. C. The plates are incubated for 2 days and, at that
time, numerous colonies could be seen growing on the agar. Some of
these colonies are picked and subcultured onto nutrient agar
plates. These apparent resistant cultures are tested for resistance
by cross streaking with the phage lysate. A line of the phage
lysate is streaked on the plate and allowed to dry. The presumptive
resistant cultures are then streaked across the phage line.
Resistant bacterial cultures show no lysis anywhere in the streak
across the phage line after overnight incubation at 30.degree. C.
The resistance to phage is then reconfirmed by plating a lawn of
the resistant culture onto a nutrient agar plate. The sensitive
strain is also plated in the same manner to serve as the positive
control. After drying, a drop of the phage lysate is plated in the
center of the plate and allowed to dry. Resistant cultures showed
no lysis in the area where the phage lysate has been placed after
incubation at 30.degree. C. for 24 hours.
4.7 Crystal Protein Compositions as Insecticides and Methods of
Use
Order Coleoptera comprises numerous beetle species including ground
beetles, reticulated beetles, skin and larder beetles, long-horned
beetles, leaf beetles, weevils, bark beetles, ladybird beetles,
soldier beetles, stag beetles, water scavenger beetles, and a host
of other beetles. A brief taxonomy of the Order is given at the
website http://www.ncbi.nlm.nih.gov/Taxonomy/tax.html.
Particularly important among the Coleoptera are the agricultural
pests included within the infraorders Chrysomeliformia and
Cucujiformia. Members of the infraorder Chrysomeliformia, including
the leaf beetles (Chrysomelidae) and the weevils (Curculionidae),
are particularly problematic to agriculture, and are responsible
for a variety of insect damage to crops and plants. The infraorder
Cucujiformia includes the families Coccinellidae, Cucujidae,
Lagridae, Meloidae, Rhipiphoridae, and Tenebrionidae. Within this
infraorder, members of the family Chrysomelidae (which includes the
genera Exema, Chrysomela, Oreina, Chrysolina, Leptinotarsa,
Gonioctena, Oulema, Monozia, Ophraella, Cerotoma, Diabrotica, and
Lachnaia), are well-known for their potential to destroy
agricultural crops.
As the toxins of the present invention have been shown to be
effective in combatting a variety of members of the order
Coleoptera, the inventors contemplate that the insects of many
Coleopteran genera may be controlled or eradicated using the
polypeptide compositions described herein. Likewise, the methods
described herein for generating modified polypeptides having
enhanced insect specificity may also be useful in extending the
range of the insecticidal activity of the modified polypeptides to
other insect species within, and outside of, the Order
Coleoptera.
As such, the inventors contemplate that the crystal protein
compositions disclosed herein will find particular utility as
insecticides for topical and/or systemic application to field
crops, including but not limited to rice, wheat, alfalfa, corn
(maize), soybeans, tobacco, potato, barley, canola (rapeseed),
sugarbeet, sugarcane, flax, rye, oats, cotton, sunflower; grasses,
such as pasture and turf grasses; fruits, citrus, nuts, trees,
shrubs and vegetables; as well as ornamental plants, cacti,
succulents, and the like.
Disclosed and claimed is a composition comprising an
insecticidally-effective amount of a Cry3Bb* crystal protein
composition. The composition preferably comprises the amino acid
sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ
ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,
SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID
NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ
ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46,
SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID
NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ
ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, or SEQ ID
NO:108, or biologically-functional equivalents thereof.
The insecticide composition may also comprise a Cry3Bb* crystal
protein that is encoded by a nucleic acid sequence having the
sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ
ID NO:9, SEQ ID NO:11, SEQ ID NO:13. SEQ ID NO:15, SEQ ID NO:17,
SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID
NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ
ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45,
SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID
NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ
ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99, or SEQ ID
NO:108, or, alternatively, a nucleic acid sequence which hybridizes
to the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:1, SEQ ID NO:13. SEQ ID
NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ
ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33,
SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID
NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ
ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61,
SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID
NO:99, or SEQ ID NO:107 under conditions of moderate
stringency.
The insecticidal compositions may comprise one or more B.
thuringiensis cell types, or one or more cultures of such cells, or
alternatively, a mixture of one of more B. thuringiensis cells
which express one or more of the novel crystal proteins of the
invention in combination with another insecticidal composition. In
certain aspects it may be desirable to prepare compositions which
contain a plurality of crystal proteins, either native or modified,
for treatment of one or more types of susceptible insects. The B.
thuringiensis cells of the invention can be treated prior to
formulation to prolong the insecticidal activity when the cells are
applied to the environment of the target insect(s). Such treatment
can be by chemical or physical means, or by a combination of
chemical and/or physical means, so long as the technique does not
deleteriously affect the properties of the insecticide, nor
diminish the cellular capability in protecting the insecticide.
Examples of chemical reagents are halogenating agents, particularly
halogens of atomic no. 17-80. More particularly, iodine can be used
under mild conditions and for sufficient time to achieve the
desired results. Other suitable techniques include treatment with
aldehydes, such as formaldehyde and glutaraldehyde;
anti-infectives, such as zephiran chloride; alcohols, such as
isopropyl and ethanol; various histologic fixatives, such as
Bouin's fixative and Helly's fixative (see Humason, 1967); or a
combination of physical (heat) and chemical agents that prolong the
activity of the .delta.-endotoxin produced in the cell when the
cell is applied to the environment of the target pest(s). Examples
of physical means are short wavelength radiation such as
gamma-radiation and X-radiation, freezing, UV irradiation,
lyophilization, and the like.
The inventors contemplate that any formulation methods known to
those of skill in the art may be employed using the proteins
disclosed herein to prepare such bioinsecticide compositions. It
may be desirable to formulate whole cell preparations, cell
extracts, cell suspensions, cell homogenates, cell lysates, cell
supernatants, cell filtrates, or cell pellets of a cell culture
(preferably a bacterial cell culture such as a B. thuringiensis
cell culture described in Table 3) that expresses one more cry3Bb*
DNA segments to produce the encoded Cry3Bb* protein(s) or
peptide(s). The methods for preparing such formulations are known
to those of skill in the art, and may include, e.g., desiccation,
lyophilization, homogenization, extraction, filtration,
centrifugation, sedimentation, or concentration of one or more
cultures of bacterial cells, such as B. thuringiensis cells
described in Table 3, which express the Cry3Bb* peptide(s) of
interest.
In one preferred embodiment, the bioinsecticide composition
comprises an oil flowable suspension comprising lysed or unlysed
bacterial cells, spores, or crystals which contain one or more of
the novel crystal proteins disclosed herein. Preferably the cells
are B. thuringiensis cells, however, any such bacterial host cell
expressing the novel nucleic acid segments disclosed herein and
producing a crystal protein is contemplated to be useful, such as
Bacillus spp., including B. megaterium, B. subtilis; B. cereus,
Escherichia spp., including E. coli, and/or Pseudomonas spp.,
including P. cepacia, P. aeruginosa, and P. fluorescens.
Alternatively, the oil flowable suspension may consist of a
combination of one or more of the following compositions: lysed or
unlysed bacterial cells, spores, crystals, and/or purified crystal
proteins.
In a second preferred embodiment, the bioinsecticide composition
comprises a water dispersible granule or powder. This granule or
powder may comprise lysed or unlysed bacterial cells, spores, or
crystals which contain one or more of the novel crystal proteins
disclosed herein. Preferred sources for these compositions include
bacterial cells such as B. thuringiensis cells, however, bacteria
of the genera Bacillus, Escherichia, and Pseudomonas which have
been transformed with a DNA segment disclosed herein and expressing
the crystal protein are also contemplated to be useful.
Alternatively, the granule or powder may consist of a combination
of one or more of the following compositions: lysed or unlysed
bacterial cells, spores, crystals, and/or purified crystal
proteins.
In a third important embodiment, the bioinsecticide composition
comprises a wettable powder, spray, emulsion, colloid, aqueous or
organic solution, dust, pellet, or collodial concentrate. Such a
composition may contain either unlysed or lysed bacterial cells,
spores, crystals, or cell extracts as described above, which
contain one or more of the novel crystal proteins disclosed herein.
Preferred bacterial cells are B. thuringiensis cells, however,
bacteria such as B. megaterium, B. subtilis, B. cereus, B. coli, or
Pseudomonas spp. cells transformed with a DNA segment disclosed
herein and expressing the crystal protein are also contemplated to
be useful. Such dry forms of the insecticidal compositions may be
formulated to dissolve immediately upon wetting, or alternatively,
dissolve in a controlled-release, sustained-release, or other
time-dependent manner. Alternatively, such a composition may
consist of a combination of one or more of the following
compositions: lysed or unlysed bacterial cells, spores, crystals,
and/or purified crystal proteins.
In a fourth important embodiment, the bioinsecticide composition
comprises an aqueous solution or suspension or cell culture of
lysed or unlysed bacterial cells, spores, crystals, or a mixture of
lysed or unlysed bacterial cells, spores, and/or crystals, such as
those described above which contain one or more of the novel
crystal proteins disclosed herein. Such aqueous solutions or
suspensions may be provided as a concentrated stock solution which
is diluted prior to application, or alternatively, as a diluted
solution ready-to-apply.
For these methods involving application of bacterial cells, the
cellular host containing the Crystal protein gene(s) may be grown
in any convenient nutrient medium, where the DNA construct provides
a selective advantage, providing for a selective medium so that
substantially all or all of the cells retain the B. thuringiensis
gene. These cells may then be harvested in accordance with
conventional ways. Alternatively, the cells may be treated prior to
harvesting.
When the insecticidal compositions comprise B. thuringiensis cells,
spores, and/or crystals containing the modified crystal protein(s)
of interest, such compositions may be formulated in a variety of
ways. They may be employed as wettable powders, granules or dusts,
by mixing with various inert materials, such as inorganic minerals
(phyllosilicates, carbonates, sulfates, phosphates, and the like)
or botanical materials (powdered comcobs, rice hulls, walnut
shells, and the like). The formulations may include
spreader-sticker adjuvants, stabilizing agents, other pesticidal
additives, or surfactants. Liquid formulations may be aqueous-based
or non-aqueous and employed as foams, suspensions, emulsifiable
concentrates, or the like. The ingredients may include rheological
agents, surfactants, emulsifiers, dispersants, or polymers.
Alternatively, the novel Cry3Bb-derived mutated crystal proteins
may be prepared by native or recombinant bacterial expression
systems in vitro and isolated for subsequent field application.
Such protein may be either in crude cell lysates, suspensions,
colloids, etc., or alternatively may be purified, refined,
buffered, and/or further processed, before formulating in an active
biocidal formulation. Likewise, under certain circumstances, it may
be desirable to isolate crystals and/or spores from bacterial
cultures expressing the crystal protein and apply solutions,
suspensions, or collodial preparations of such crystals and/or
spores as the active bioinsecticidal composition.
Another important aspect of the invention is a method of
controlling coleopteran insects which are susceptible to the novel
compositions disclosed herein. Such a method generally comprises
contacting the insect or insect population, colony, etc., with an
insecticidally-effective amount of a Cry3Bb* crystal protein
composition. The method may utilize Cry3Bb* crystal proteins such
as those disclosed in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID
NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ
ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26,
SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID
NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ
ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54,
SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID
NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, or
SEQ ID NO:108, or biologically functional equivalents thereof.
Alternatively, the method may utilize one or more Cry3Bb* crystal
proteins which are encoded by the nucleic acid sequences of SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ
ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,
SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID
NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ
ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57,
SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID
NO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ ID NO:101, or SEQ ID NO:107,
or by one or more nucleic acid sequences which hybridize to the
subsequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ
ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35,
SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID
NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ
ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63,
SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ ID
NO:101, or SEQ ID NO:107, under conditions of moderate, or higher,
stringency. The methods for identifying sequences which hybridize
to those disclosed under conditions of moderate or higher
stringency are well-known to those of skill in the art, and are
discussed herein.
Regardless of the method of application, the amount of the active
component(s) are applied at an insecticidally-effective amount,
which will vary depending on such factors as, for example, the
specific colopteran insects to be controlled, the specific plant or
crop to be treated, the environmental conditions, and the method,
rate, and quantity of application of the insecticidally-active
composition.
The insecticide compositions described may be made by formulating
either the bacterial cell, crystal and/or spore suspension, or
isolated protein component with the desired
agriculturally-acceptable carrier. The compositions may be
formulated prior to administration in an appropriate means such as
lyophilized, freeze-dried, dessicated, or in an aqueous carrier,
medium or suitable diluents, such as saline or other buffer. The
formulated compositions may be in the form of a dust or granular
material, or a suspension in oil (vegetable or mineral), or water
or oil/water emulsions, or as a wettable powder, or in combination
with any other carrier material suitable for agricultural
application. Suitable agricultural carriers can be solid or liquid
and are well known in the art. The term "agriculturally-acceptable
carrier" covers all adjuvants, e.g., inert components, dispersants,
surfactants, tackifiers, binders, etc. that are ordinarily used in
insecticide formulation technology; these are well known to those
skilled in the insecticide formulation. The formulations may be
mixed with one or more solid or liquid adjuvants and prepared by
various means, e.g. by homogeneously mixing, blending and/or
grinding the insecticidal composition with suitable adjuvants using
conventional formulation techniques.
The insecticidal compositions of this invention are applied to the
environment of the target coleopteran insect, typically onto the
foliage of the plant or crop to be protected, by conventional
methods, preferably by spraying. The strength and duration of
insecticidal application will be set with regard to conditions
specific to the particular pest(s), crop(s) to be treated with
particular environmental conditions. The proportional ratio of
active ingredient to carrier will naturally depend on the chemical
nature, solubility, and stability of the insecticidal composition,
as well as the particular formulation contemplated.
Other application techniques, e.g., dusting, sprinkling, soaking,
soil injection, soil tilling, seed coating, seedling coating,
spraying, aerating, misting, atomizing, and the like, are also
feasible and may be required under certain circumstances such as
e.g., insects that cause root or stalk infestation, or for
application to delicate vegetation or ornamental plants. These
application procedures are also well-known to those of skill in the
art.
The insecticidal composition of the invention may be employed in
the method of the invention singly or in combination with other
compounds, including and not limited to other pesticides. The
method of the invention may also be used in conjunction with other
treatments such as surfactants, detergents, polymers or
time-release formulations. The insecticidal compositions of the
present invention may be formulated for either systemic or topical
use.
The concentration of insecticidal composition which is used for
environmental, systemic, or foliar application will vary widely
depending upon the nature of the particular formulation, means of
application, environmental conditions, and degree of biocidal
activity. Typically, the bioinsecticidal composition will be
present in the applied formulation at a concentration of at least
about 1% by weight and may be up to and including about 99% by
weight. Dry formulations of the compositions may be from about 1%
to about 99% or more by weight of the composition, while liquid
formulations may generally comprise from about 1% to about 99% or
more of the active ingredient by weight. Formulations which
comprise intact bacterial cells will generally contain from about
10.sup.4 to about 10.sup.2 cells/mg.
The insecticidal formulation may be administered to a particular
plant or target area in one or more applications as needed, with a
typical field application rate per hectare ranging on the order of
from about 1 g to about 1 kg, 2 kg, 5, kg, or more of active
ingredient.
4.8 Nucleic Acid Segments as Hybridization Probes and Primers
In addition to their use in directing the expression of crystal
proteins or peptides of the present invention, the nucleic acid
sequences contemplated herein also have a variety of other uses.
For example, they also have utility as probes or primers in nucleic
acid hybridization embodiments. As such, it is contemplated that
nucleic acid segments that comprise a sequence region that consists
of at least a 14 nucleotide long contiguous sequence that has the
same sequence as, or is complementary to, a 14 nucleotide long
contiguous DNA segment of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13. SEQ ID NO:15,
SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID
NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ
ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43,
SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID
NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ
ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99,
SEQ ID NO:101, or SEQ ID NO:107 will find particular utility.
Longer contiguous identical or complementary sequences, e.g., those
of about 20, 30, 40, 50, 100, 200, 500, 1000, 2000, 5000, 10000
etc. (including all intermediate lengths and up to and including
full-length sequences will also be of use in certain
embodiments.
The ability of such nucleic acid probes to specifically hybridize
to crystal protein-encoding sequences will enable them to be of use
in detecting the presence of complementary sequences in a given
sample. However, other uses are envisioned, including the use of
the sequence information for the preparation of mutant species
primers, or primers for use in preparing other genetic
constructions.
Nucleic acid molecules having sequence regions consisting of
contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of
100-200 nucleotides or so, identical or complementary to DNA
sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ
ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35,
SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID
NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ
ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63,
SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ ID
NO:101, or SEQ ID NO:107 are particularly contemplated as
hybridization probes for use in e.g., Southern and Northern
blotting. Smaller fragments will generally find use in
hybridization embodiments, wherein the length of the contiguous
complementary region may be varied, such as between about 10-14 and
about 100 or 200 nucleotides, but larger contiguous complementary
stretches may be used, according to the length complementary
sequences one wishes to detect.
The use of a hybridization probe of about 14 nucleotides in length
allows the formulation of a duplex molecule that is both stable and
selective. Molecules having contiguous complementary sequences over
stretches greater than 14 bases in length are generally preferred,
though, in order to increase stability and selectivity of the
hybrid, and thereby improve the quality and degree of specific
hybrid molecules obtained. One will generally prefer to design
nucleic acid molecules having gene-complementary stretches of 15 to
20 contiguous nucleotides, or even longer where desired.
Of course, fragments may also be obtained by other techniques such
as, e.g., by mechanical shearing or by restriction enzyme
digestion. Small nucleic acid segments or fragments may be readily
prepared by, for example, directly synthesizing the fragment by
chemical means, as is commonly practiced using an automated
oligonucleotide synthesizer. Also, fragments may be obtained by
application of nucleic acid reproduction technology, such as the
PCR.TM. technology of U.S. Pat. No. 4,683,195 and 4,683,202 (each
incorporated herein by reference), by introducing selected
sequences into recombinant vectors for recombinant production, and
by other recombinant DNA techniques generally known to those of
skill in the art of molecular biology.
Accordingly, the nucleotide sequences of the invention may be used
for their ability to selectively form duplex molecules with
complementary stretches of DNA fragments. Depending on the
application envisioned, one will desire to employ varying
conditions of hybridization to achieve varying degrees of
selectively of probe toward target sequence. For applications
requiring high selectivity, one will typically desire to employ
relatively stringent conditions to form the hybrids, e.g., one will
select relatively low salt and/or high temperature conditions, such
as provided by about 0.02 M to about 0.15 M NaCl at temperatures of
about 50.degree. C. to about 70.degree. C. Such selective
conditions tolerate little, if any, mismatch between the probe and
the template or target strand, and would be particularly suitable
for isolating crystal protein-encoding DNA segments. Detection of
DNA segments via hybridization is well-known to those of skill in
the art, and the teachings of U.S. Pat. Nos. 4,965,188 and
5,176,995 (each incorporated herein by reference) are exemplary of
the methods of hybridization analyses. Teachings such as those
found in the texts of Maloy et al., 1994; Segal 1976; Prokop, 1991;
and Kuby, 1994, are particularly relevant.
Of course, for some applications, for example, where one desires to
prepare mutants employing a mutant primer strand hybridized to an
underlying template or where one seeks to isolate crystal
protein-encoding sequences from related species, functional
equivalents, or the like, less stringent hybridization conditions
will typically be needed in order to allow formation of the
heteroduplex. In these circumstances, one may desire to employ
conditions such as about 0.15 M to about 0.9 M salt, at
temperatures ranging from about 20.degree. C. to about 55.degree.
C. Cross-hybridizing species can thereby be readily identified as
positively hybridizing signals with respect to control
hybridizations. In any case, it is generally appreciated that
conditions can be rendered more stringent by the addition of
increasing amounts of formamide, which serves to destabilize the
hybrid duplex in the same manner as increased temperature. Thus,
hybridization conditions can be readily manipulated, and thus will
generally be a method of choice depending on the desired
results.
In certain embodiments, it will be advantageous to employ nucleic
acid sequences of the present invention in combination with an
appropriate means, such as a label, for determining hybridization.
A wide variety of appropriate indicator means are known in the art,
including fluorescent, radioactive, enzymatic or other ligands,
such as avidin/biotin, which are capable of giving a detectable
signal. In preferred embodiments, one will likely desire to employ
a fluorescent label or an enzyme tag, such as urease, alkaline
phosphatase or peroxidase, instead of radioactive or other
environmental undesirable reagents. In the case of enzyme tags,
calorimetric indicator substrates are known that can be employed to
provide a means visible to the human eye or spectrophotometrically,
to identify specific hybridization with complementary nucleic
acid-containing samples.
In general, it is envisioned that the hybridization probes
described herein will be useful both as reagents in solution
hybridization as well as in embodiments employing a solid phase. In
embodiments involving a solid phase, the test DNA (or RNA) is
adsorbed or otherwise affixed to a selected matrix or surface. This
fixed, single-stranded nucleic acid is then subjected to specific
hybridization with selected probes under desired conditions. The
selected conditions will depend on the particular circumstances
based on the particular criteria required (depending, for example,
on the G+C content, type of target nucleic acid, source of nucleic
acid, size of hybridization probe, etc.) Following washing of the
hybridized surface so as to remove nonspecifically bound probe
molecules, specific hybridization is detected, or even quantitated,
by means of the label.
4.9 Characteristics of Modified Cry3 .delta.-endotoxins
The present invention provides novel polypeptides that define a
whole or a portion of a B. thuringiensis cry3Bb.60, cry3Bb.11221,
cry3Bb.11222, cry3Bb.11223, cry3Bb.11224, cry3Bb.11225,
cry3Bb.11226, cry3Bb.11227, cry3Bb.11228, cry3Bb.11229,
cry3Bb.11230, cry3Bb.11231, cry3Bb.11232, cry3Bb.11233,
cry3Bb.11234, cry3Bb.11235, cry3Bb.11236, cry3Bb.11237,
cry3Bb.11238, cry3Bb.11239, cry3Bb.11241, cry3Bb.11242,
cry3Bb.11032, cry3Bb.11035, cry3Bb.11036, cry3Bb.11046,
cry3Bb.11048, cry3Bb.11051, cry3Bb.11057, cry3Bb.11058,
cry3Bb.11081, cry3Bb.11082, cry3Bb.11083, cry3Bb.11084,
cry3Bb.11095, and cry3Bb.11098-encoded crystal protein.
4.10 Crystal Protein Nomenclature
The inventors arbitrarily assigned the designations Cry3Bb.60,
Cry3Bb.11221, Cry3Bb.11222, Cry3Bb.11223, Cry3Bb.11224,
Cry3Bb.11225, Cry3Bb.11226, Cry3Bb.11227, Cry3Bb.11228,
Cry3Bb.11229, Cry3Bb.11230, Cry3Bb.11231, Cry3Bb.11232,
Cry3Bb.11233, Cry3Bb.11234, Cry3Bb.11235, Cry3Bb.11236,
Cry3Bb.11237, Cry3Bb.11238, Cry3Bb.11239, Cry3Bb.11241,
Cry3Bb.11242, Cry3Bb.11032, Cry3Bb.11035, Cry3Bb.11036,
Cry3Bb.11046, Cry3Bb.11048, Cry3Bb.11051, Cry3Bb.11057,
Cry3Bb.11058, Cry3Bb.11081, Cry3Bb.11082, Cry3Bb.11083,
Cry3Bb.11084, Cry3Bb.11095, and Cry3Bb.11098 to the novel proteins
of the invention.
Likewise, the arbitrary designations of Cry3Bb.60 cry3Bb.11221,
cry3Bb.11222, cry3Bb.11223, cry3Bb.11224, cry3Bb.11225,
cry3Bb.11226, cry3Bb.11227, cry3Bb.11228, cry3Bb.11229,
cry3Bb.11230, cry3Bb.11231, cry3Bb.11232, cry3Bb.11233,
cry3Bb.11234, cry3Bb.11235, cry3Bb.11236, cry3Bb.11237,
cry3Bb.11238, cry3Bb.11239, cry3Bb.11241, cry3Bb.11242,
cry3Bb.11032, cry3Bb.11035, cry3Bb.11036, cry3Bb.11046,
cry3Bb.11048, cry3Bb.11051, cry3Bb.11057, cry3Bb.11058,
cry3Bb.11081, cry3Bb.11082, cry3Bb.11083, cry3Bb.11084,
cry3Bb.11095 and Cry3Bb.11098 have been assigned to the novel
nucleic acid sequences which encode these polypeptides,
respectively. While format assignment of gene and protein
designations based on the revised nomenclature of crystal protein
endotoxins (Table 1) may be made by the committee on the
nomenclature of B. thuringiensis, any re-designations of the
compositions of the present invention are also contemplated to be
fully within the scope of the present disclosure.
4.11 Transformed Host Cells and Transgenic Plants
A bacterium, a yeast cell, or a plant cell or a plant transformed
with an expression vector of the present invention is also
contemplated. A transgenic bacterium, yeast cell, plant cell or
plant derived from such a transformed or transgenic cell is also
one aspect of the invention.
Such transformed host cells are often desirable for use in the
production of endotoxins and for expression of the various DNA gene
constructs disclosed herein. In some aspects of the invention, it
is often desirable to modulate, regulate, or otherwise control the
expression of the gene segments disclosed herein. Such methods are
routine to those of skill in the molecular genetic arts. Typically,
when increased or over-expression of a particular gene is desired,
various manipulations may be employed for enhancing the expression
of the messenger RNA, particularly by using an active promoter, as
well as by employing sequences, which enhance the stability of the
messenger RNA in the particular transformed host cell.
Typically, the initiation and translational termination region will
involve stop codon(s), a terminator region, and optionally, a
polyadenylation signal. In the direction of transcription, namely
in the 5' to 3' direction of the coding or sense sequence, the
construct will involve the transcriptional regulatory region, if
any, and the promoter, where the regulatory region may be either 5'
or 3' of the promoter, the ribosomal binding site, the initiation
codon, the structural gene having an open reading frame in phase
with the initiation codon, the stop codon(s), the polyadenylation
signal sequence, if any, and the terminator region. This sequence
as a double strand may be used by itself for transformation of a
microorganism host, but will usually be included with a DNA
sequence involving a marker, where the second DNA sequence may be
joined to the .delta.-endotoxin expression construct during
introduction of the DNA into the host.
By a marker is intended a structural gene which provides for
selection of those hosts which have bene modified or transformed.
The marker will normally provide for selective advantage, for
example, providing for biocide resistance, e.g., resistance to
antibiotics or heavy metals; complementation, so as to provide
prototropy to an auxotrophic host, or the like. Preferably,
complementation is employed, so that the modified host may not only
be selected, but may also be competitive in the field. One or more
markers may be employed in the development of the constructs, as
well as for modifying the host. The organisms may be further
modified by providing for a competitive advantage against other
wild-type microorganisms in the field. For example, genes
expressing metal chelating agents, e.g., siderophores, may be
introduced into the host along with the structural gene expressing
the .delta.-endotoxin. In this manner, the enhanced expression of a
siderophore may provide for a competitive advantage for the
.delta.-endotoxin-producing host, so that it may effectively
compete with the wild-type microorganisms and stably occupy a niche
in the environment.
Where no functional replication system is present, the construct
will also include a sequence of at least 50 basepairs (bp),
preferably at least about 100 bp, and usually not more than about
1000 bp of a sequence homologous with a sequence in the host. In
this way, the probability of legitimate recombination is enhanced,
so that the gene will be integrated into the host and stably
maintained by the host. Desirably, the .delta.-endotoxin gene will
be in close proximity to the gene providing for complementation as
well as the gene providing for the competitive advantage.
Therefore, in the event that a .delta.-endotoxin gene is lost, the
resulting organism will be likely to also lose the complementing
gene and/or the gene providing for the competitive advantage, so
that it will be unable to compete in the environment with the gene
retaining the intact construct.
The crystal protein-encoding gene can be introduced between the
transcriptional and translational initiation region and the
transcriptional and translational termination region, so as to be
under the regulatory control of the initiation region. This
construct will be included in a plasmid, which will include at
least one replication system, but may include more than one, where
one replication system is employed for cloning during the
development of the plasmid and the second replication system is
necessary for functioning in the ultimate host. In addition, one or
more markers may be present, which have been described previously.
Where integration is desired, the plasmid will desirably include a
sequence homologous with the host genome.
The transformants can be isolated in accordance with conventional
ways, usually employing a selection technique, which allows for
selection of the desired organism as against unmodified organisms
or transferring organisms, when present. The transformants then can
be tested for pesticidal activity.
Suitable host cells, where the pesticide-containing cells will be
treated to prolong the activity of the .delta.-endotoxin in the
cell when the then treated cell is applied to the environment of
the target pest(s), may include either prokaryotes or eukaryotes,
normally being limited to those cells which do not produce
substances toxic to higher organisms, such as mammals. However,
organisms which produce substances toxic to higher organisms could
be used, where the .delta.-endotoxin is unstable or the level of
application sufficiently low as to avoid any possibility of
toxicity to a mammalian host. As hosts, of particular interest will
be the prokaryotes and the lower eukaryotes, such as fungi,
illustrative prokaryotes, both Gramm-negative and -positive,
include Enterobacteriacae, such as Escherichia, Erwinia, Shigella,
Salmonella, and Proteus; Bacillaceae; Rhizobicae, such as
Rhizobium; Spirillaceae, such as photobacterium, Zymomonas,
Serratia, Aeromonoas, Vibrio, Desulfovibdo, Spirillum;
Lactobacillaceae; phylloplane organisms such as members of the
Pseudomonadaceae (including Pseudomonas spp. and Acetobacter spp.);
Azotobacteraceae and Nitrobacteraceae, Flavobacterium spp.; members
of the Bacillaceae such as Lactobacillus spp., Bifidobacterium, and
Bacillus spp., and the like. Particularly preferred host cells
include Pseudomonas aeruginosa, Pseudomonas fluorescens, Bacillus
thuringiensis, Escherichia coli, Bacillus subtilis, and the
like.
Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes,
which includes yeast, such as Schizosaccharomyces; and
Basidiomycetes, Rhodotorula, Aureobasidium, Sporobotomyces,
Saccharomyces spp., and Sporobolomyces spp.
Characteristics of particular interest in selecting a host cell for
purposes of production include ease of introducing the
.delta.-endotoxin gene into the host, availability of expression
systems, efficiency of expression, stability of the pesticide in
the host, and the presence of auxiliary genetic capabilities.
Characteristics of interest for use as a pesticide microcapsule
include protective qualities for the pesticide, such as thick cell
walls, pigmentation, and intracellular packaging or formation of
inclusion bodies; leaf affinity; lack of mammalian toxicity;
attractiveness to pests for ingestion; ease of killing and fixing
without damage to the .delta.-endotoxin; and the like. Other
considerations include case of formulation and handling, economics,
storage stability, and the like.
The cell will usually be intact and be substantially in the
proliferative form when treated, rather than in a spore form,
although in some instance spores may be employed. Treatment of the
recombinant microbial cell can be done as disclosed infra. The
treated cells generally will have enhanced structural stability
which will enhance resistance to environmental conditions.
Genes or other nucleic acid segments, as disclosed herein, can be
inserted into host cells using a variety of techniques which are
well known in the art. For example, a large number of cloning
vectors comprising a replication system in E. coli and a marker
that permits selection of the transformed cells are available for
preparation for the insertion of foreign genes into higher
organisms, including plants. The vectors comprise, for example,
pBR322, pUC series, M13mp series, pACY184, etc. Accordingly, the
sequence coding for the .delta.-endotoxin can be inserted into the
vector at a suitable restriction site. The resulting plasmid is
used for transformation into E. coli. The E. coli cells are
cultivated in a suitable nutrient medium, then harvested and lysed.
The plasmid is recovered. Sequence analysis, restriction analysis,
electrophoresis, and other biochemical-molecular biological methods
are generally carried out as methods of analysis. After each
manipulation, the DNA sequence used can be cleaved and joined to
the next DNA sequence. Each plasmid sequence can be cloned in the
same or other plasmids. Depending on the method of inserting
desired genes into the plant, other DNA sequences may be
necessary.
Methods for DNA transformation of plant cells include
Agrobacterium-mediated plant transformation, protoplast
transformation, gene transfer into pollen, injection into
reproductive organs, injection into immature embryos and particle
bombardment. Each of these methods has distinct advantages and
disadvantages. Thus, one particular method of introducing genes
into a particular plant strain may not necessarily be the most
effective for another plant strain, but it is well known which
methods are useful for a particular plant strain.
Suitable methods are believed to include virtually any method by
which DNA can be introduced into a cell, such as by Agrobacterium
infection, direct delivery of DNA such as, for example, by
PEG-mediated transformation of protoplasts (Omiruelleh et al.,
1993), by desiccation/inhibition-mediated DNA uptake, by
electroporation, by agitation with silicon carbide fibers, by
acceleration of DNA coated particles, etc. In certain embodiments,
acceleration methods are preferred and include, for example,
microprojectile bombardment and the like.
Technology for introduction of DAN into cells is well-known to
those of skill in the art. Four general methods for delivering a
gene into cells have been described: (1) chemical methods (Graham
and van der Eb, 1973; Zatlouka et al., 1992); (2) physical methods
such as microinjection (Capecchi, 1980), electroporation (Wong and
Neumann, 1982; Fromm et al., 1985) and the gene gun (Johnston and
Tang, 1994; Fynan et al., 1993); (3) viral vectors (Clapp, 1993; Lu
et al., 1993; Eglitis and Anderson, 1988; Eglitis et al., 1988);
and (4) receptor-mediated mechanisms (Curiel et al., 1991; 1992;
Wagner et al., 1992).
A large number of techniques are available for inserting DNA into a
plant host cell. Those techniques include transformation with T-DNA
using Agrobacterium tumefaciens or Agrobacterium rhizogenes as
transformation agent, fusion, injection, or electroporation as well
as other possible methods. If agrobacteria are used for the
transformation, the DNA to be inserted has to be cloned into
special plasmids, namely either into an intermediate vector or into
a binary vector. The intermediate vectors can be integrated into
the Ti or Ri plasmid by homologous recombination owing to sequences
that are homologous to sequences in the T-DNA. The Ti or Ri plasmid
also comprises the vir region necessary for the transfer of the
T-DNA.
Intermediate vectors cannot replicate themselves in agrobacteria.
The intermediate vector can be transferred into Agrobacterium
tumefaciens by means of a helper plasmid (conjugation). Binary
vectors can replicate themselves both in E. coli and in
agrobacteria. They comprise a selection marker gene and a linker or
polylinker which are framed by the right and left T-DNA border
regions. They can be transformed directly into agrobacteria
(Holsters et al., 1978). The agrobacterium used as host cell is to
comprise a plasmid carrying a vir region. The vir region is
necessary for the transfer of the T-DNA into the plant cell.
Additional t-DNA may be contained. The bacterium so transformed is
used for the transformation of plant cells. Plant explants can
advantageously be cultivated with Agrobacterium lumefaciens or
Agrobacterium rhizogenes for the transfer of the DNA into the plant
cell. Whole plants can then be regenerated from the infected plant
material (for example, pieces of leaf, segments of stalk, roots,
but also protoplasts or suspension-cultivated cells) in a suitable
medium, which may contain antibiotics or biocides for selection.
The plants so obtained can then be tested for the presence of the
inserted DNA. No special demands are made of the plasmids in the
case of injection and electroporation. It is possible to use
ordinary plasmids, such as, for example, pUC derivatives. If, for
example, the Ti or Ri plasmid is used for the transformation of the
plant cell, then at least the right border, but often the right and
the left border of the Ti or Ri plasmid T-DNA, has to be joined as
the flanking region of the genes to be inserted. The use of T-DNA
for the transformation of plant cells has been intensively
researched and sufficiently described in Eur. Pat. Appl. No. EP 120
516; Hockema (1985); An et al., 1985, Herrera-Estrella et al.,
(1983), Bevan et al., (1983), and Klee et al., (1985).
A particularly useful Ti plasmid cassette vector for transformation
of dicotyledonous plants consists of the enhanced CaMV35S promoter
(EN35S) and the 3' end including polyadenylation signals from a
soybean gene encoding the .alpha.'-subunit of .beta.-conglycinin.
Between these two elements is a multilinker containing multiple
restriction sites for the insertion of genes of interest.
The vector preferably contains a segment of pBR322 which provides
an origin of replication in E. coli and a region for homologous
recombination with the disarmed T-DNA in Agrobacterium strain ACO;
the oriV region from the broad host range plasmid RK1; the
streptomycin/spectinomycin resistance gene form Tn7; and a chimeric
NPTII gene, containing the CaMV35S promoter and the nopaline
synthase (NOS) 3' end, which provides kinamycin resistance in
transformed plant cells.
Optionally, the enhanced CaMV35S promoter may be replaced with the
1.5 kb manopine synthase (MA) promoter (Velten et al., 1984). After
incorporation of a DNA construct into the vector, it is introduced
into A. tumefaciens strain ACO which contains a disarmed Ti
plasmid. Cointegrate Ti plasmid vectors are selected and
subsequentially may be used to transform a dicotyledonous
plant.
A. tumefaciens ACO is a disarmed strain similar to pTiB6SE
described by Fraley et al. (1985). For construction of aCO the
starting Agrobacterium strain was the strain A208 which contains a
nopaline-type Ti plasmid. The Ti plasmid was disarmed in a manner
similar to that described by Fraley et al. (1985) so that
essentially all of the native T-DNA was removed except for the leaf
border and a few hundred base pairs of T-DNA inside the left
border. The remainder of the T-DNA extending to a point just beyond
the right border was replaced with a novel piece of DNA including
(from left to right) a segment of pBR322, the oriV region from
plasmid RK2, and the kanamycin resistance gene from Tn601. The
pBR322 and oriV segments are similar to those segments and provide
a region of homology for cointegrated formation.
Once the inserted DNA has been integrated in the genome, it is
relatively stable there and, as a rule, does not come out again. It
normally contains a selection marker that confers on the
transformated plant cells resistance to a biocide or an antibiotic,
such as kinamycin, G 418, bleomycin, hygromycin, or
chloramphenicol, inter alia. The individually employed marker
should accordingly permit the selection of transformed cells rather
than cells that do not contain the inserted DNA.
4.11.1 Electroporation
The application of brief, high-voltage electric pulses to a variety
of animal and plant cells leads to the formation of nanometer-sized
pores in the plasma membrane. DNA is taken directly into the cell
cytoplasm either through these pores or as a consequence of the
redistribution of membrane components that accompanies closure of
the pores. Electroporation can be extremely efficient and can be
used both for transient expression of clones genes and for
establishment of cell lines that carry integrated copies of the
gene of interest. Electroporation, in contrast to calcium
phosphate-mediated transfection and protoplast fusion, frequently
gives rise to cell lines that carry one, or at most a few,
integrated copies of the foreign DNA.
The introduction of DNA by means of electroporation, is well-known
to those of skill in the art. In this method, certain cell
wall-degrading enzymes, such as pectin-degrading enzymes, are
employed to render the target recipient cells more susceptible to
transformation by electroporation than untreated cells.
Alternatively, recipient cells are made more susceptible to
transformation, by mechanical wounding. To effect transformation by
electroporation one may employ either friable tissues such as a
suspension culture of cells, or embryogenic callus, or
alternatively, one may transform immature embryos or other
organized tissues directly. One would partially degrade the cell
walls of the chosen cells by exposing them to pectin-degrading
enzymes (pectolyases) or mechanical wounding in a controlled
manner. Such cells would then be recipient to DNA transfer by
electroporation, which may be carried out at this stage, and
transformed cells then identified by a suitable selection or
screening protocol dependent on the nature of the newly
incorporated DNA.
4.11.2 Microprojectile Bombardment
A further advantageous method for delivering transforming DNA
segments to plant cells is microprojectile bombardment. In this
method, particles may be coated with nucleic acids and delivered
into cells by a propelling force. Exemplary particles include those
comprised of tungsten, gold, platinum, and the like.
An advantage of microprojectile bombardment, in addition to it
being an effective means of reproducibly stably transforming
monocots, is that neither the isolation of protoplasts (Cristou et
al., 1988) nor the susceptibility to Agrobacterium infection is
required. An illustrative embodiment of a method for delivering DNA
into maize cells by acceleration is a Biolistics Particle Delivery
System, which can be used to propel particles coated with DNA or
cells through a screen, such as a stainless steel or Nytex screen,
onto a filter surface covered with corn cells cultured in
suspension. The screen disperses the particles so that they are not
delivered to the recipient cells in large aggregates. It is
believed that a screen intervening between the projectile apparatus
and the cells to be bombarded reduces the size of projectiles
aggregate and may contribute to a higher frequency of
transformation by reducing damage inflicted on the recipient cells
by projectiles that are too large.
For the bombardment, cells in suspension are preferably
concentrated on filters or solid culture medium. Alternatively,
immature embryos or other target cells may be arranged on solid
culture medium. The cells to be bombarded are positioned at an
application distance below the macroprojectile stopping plate. If
desired, one or more screens are also positioned between the
acceleration device and the cells to be bombarded. Through the use
of techniques set forth herein one may obtain up to 1000 or more
foci of cells transiently expressing a marker gene. The number of
cells in a focus which express the exogenous gene product 48 hours
post-bombardment often range from 1 to 10 and average 1 to 3.
In bombardment transformation, one may optimize the prebombardment
culturing conditions and the bombardment parameters to yield the
maximum numbers of stable transformants. Both the physical and
biological parameters for bombardment are important in this
technology. Physical factors are those that involve manipulating
the DNA/microprojectile precipitate or those that affect the flight
and velocity of either the macro or microprojectiles. Biological
factors include all steps involved in manipulation of cells before
and immediately after bombardment, the osmotic adjustment of target
cells to help alleviate the trauma associated with bombardment, and
also the nature of the transforming DNA, such as linearized DNA or
intact supercoiled plasmids. It is believed that the
pre-bombardment manipulations are especially important for
successful transformation of immature embryos.
Accordingly, it is contemplated that one may wish to adjust various
of the bombardment parameters in small scale studies to fully
optimize the conditions. One may particularly wish to adjust
physical parameters such as gap distance, flight distance, tissue
distance, and helium pressure. One may also minimize the trauma
reduction factors (TRFs) by modifying conditions which influence
the physiological state of the recipient cells and which may
therefore influence transformation and integration efficiencies.
For example, the osmotic state, tissue hydration and the subculture
stage or cell cycle of the recipient cells may be adjusted for
optimum transformation. The execution of other routine adjustments
will be known to those of skill in the art in light of the present
disclosure.
4.11.3 Agrobacterium-mediated Transfer
Agrobacterium-mediated transfer is a widely applicable system for
introducing genes into plant cells because the DNA can be
introduced into whole plant tissue, thereby bypassing the need for
regeneration of an intact plant from a protoplast. The use of
Agrobacterium-mediated plant integrating vectors to introduce DNA
into plant cells is well known in the art. See, for example, the
methods described (Fraley et al, 1985; Rogers et al., 1987).
Further, the integration of th Ti-DNA is a relatively precise
process resulting in few rearrangements. The region of DNA to be
transferred is defined by the border sequences, and intervening DNA
is usually inserted into the plant genome as described (Spielmann
et al., 1986; Jorgensen et al., 1987).
Modern Agrobacterium transformation vectors are capable of
replication E. coli as well as Agrobacterium, allowing for
convenient manipulations as described (Klee et al., 1985).
Moreover, recent technological advances in vectors for
Agrobacterium-mediated gene transfer have improved the arrangement
of genes and restriction sites in the vectors to facilitate
construction of vectors capable of expressing various polypeptide
coding genes. The vectors described (Rogers et al., 1987), have
convenient multi-linker regions flanked by a promoter and a
polyadenylation site for direct expression of inserted polypeptide
coding genes and are suitable for present purposes. In addition,
Agrobacterium containing both armed and disarmed Ti genes can be
used for the transformations. In those plant strains where
Agrobacterium-mediated transformation is efficient, it is the
method of choice because of the facile and defined nature of the
gene transfer.
Agrobacterium-mediate transformation of leaf disks and other
tissues such as cotyledons and bypocotyls appears to be limited to
plants that Agrobacterium naturally infects. Agrobacterium-mediated
transformation to most efficient in dicotyledonoous plants. Few
monocots appear to be natural hosts for Agrobacterium, although
transgenic plants have been produced in asparagus using
Agrobacterium vectors as described (Bytebier et al., 1987).
Therefore, commercially important cereal grains such as rice, corn,
and wheat must usually be transformed using alternative methods.
However, as mentioned above, the transformation of asparagus using
Agrobacterium can also be achieved (see, for example, Bytebier et
al., 1987).
A transgenic plant formed using Agrobacterium transformation
methods typically contains a single gene on one chromosome. Such
transgenic plants can be referred to as being heterozygous for the
added gene. However, inasmuch as use of the word "heterozygous"
usually implies the presence of a complementary gene at the same
locus of the second chromosome of a pair of chromosomes, and there
is no such gene in a plant containing one added gene, as here, it
is believed that a more accurate name for such a plant is an
independent segregant, because the added, exogenous gene segregates
independently during mitosis and mesiosis.
More preferred is a transgenic plant that is homozygous for the
added structural gene; i.e., a transgenic plant that contains two
added genes, one gene at the same locus on each chromosome of a
chromosome pair. A homozygous transgenic plant can be obtained by
sexually mating (selfing) an independent segregant transgenic plant
that contains a single added gene, germinating some of the seed
produced and analyzing the resulting plants produced for enhanced
carboxylase activity relative to a control (native, non-transgenic)
or an independent segregant transgenic plant.
It is to be understood that two different transgenic plants can
also be mated to produce offspring that contain two independently
segregating added, exogenous genes. Selfing of appropriate progeny
can produce plants that are homozygous for both added, exogenous
genes that encode a polypeptide of interest. Back-crossing to a
parental plant and out-crossing with a non-transgenic plant are
also contemplated.
Transformation of plant protoplasts can be achieved using methods
based on calcium phosphate precipitation, polyethylene glycol
treatment, electroporation, and combination of these treatments
(see, e.g., Potrykus et al., 1985; Lorz et al., 1985; Fromm et al.,
1985; Uchimiya et al., 1986; Callis et al., 1987; Marcotte et al.,
1988).
Application of these systems to different plant strains depends
upon the ability to regenerate that particular plant strain from
protoplasts. Illustrative methods for the regeneration of cereals
from protoplasts are described (Fijimura et al., 1985; Toriyama et
al., 1986; yamada et al., 1986; Abdullab et al., 1986).
To transform plant strains that cannot be successfully regenerated
from protoplasts, other ways to introduce DNA into intact cells or
tissues can be utilized. For example, regeneration of cereals from
immature embryos or explants can be effected as described
(Vasil,1988). In addition, "particle gun" or high-velocity
microprojectile technology can be utilized (Vasil, 1992).
Using that latter technology, DAN is carried through the cell wall
and into the cytoplasm on the surface of small metal particles as
described (Klein et al., 1987; Klein et al., 1988; McCabe et al.,
1988). The metal particles penetrate through several layers of
cells and this allow the transformation of cells within tissue
explants.
4.11.4 Gene Expression in Plants
Although great progress has been made in recent years with respect
to preparation of transgenic plants which express bacterial
proteins such as B. thuringiensis crystal proteins, the results of
expressing native bacterial genes in plants are often
disappointing. Unlike microbial genetics, little was known by early
plant geneticists about the factors which afforded heterologous
expression of foreign genes in plants. In recent years, however,
several potential factors have been implicated as responsible in
varying degrees for the level of protein expression from a
particular coding sequence. For example, scientists now know that
maintaining a significant level of a particular mRNA in the cell is
indeed a critical factor. Unfortunately, the causes for low steady
state levels of mRNA encoding foreign proteins are many. First,
full length RNA synthesis may not occur at a high frequency. This
could, for example, be caused by the premature termination of RNA
during transcription or due to unexpected mRNA processing during
transcription. Second, full length RNA may be produced in the plant
cell, but then processed (splicing, polyA addition) in the nucleus
in a fashion that creates a nonfunctional mRNA. If the RNA is not
properly synthesized, terminated and polyadenylated, it cannot move
to the cytoplasm for translation. Similarly, in the cytoplasm, if
mRNAs have reduced half lives (which are determined by their
primary or secondary sequence) insufficient protein product will be
produced. In addition, there is an effect, whose magnitude is
uncertain, or translational efficiency on mRNA half-life. In
addition, every RNA molecule folds into a particular structure, or
perhaps family of structures, which is determined by its sequence.
The particular structure of any RNA might lead to greater or lesser
stability in the cytoplasm. Structure per se is probably also a
determinant of mRNA processing in the nucleus. Unfortunately, it is
impossible to predict, and nearly impossible to determine, the
structure of any RNA (except for tRNA) in vitro or in vivo.
However, it is likely that dramatically changing the sequence of an
RNA will have a large effect on its folded structure It is likely
that structure per se or particular structural features also have a
role in determining RNA stability.
To overcome these limitations in foreign gene expression,
researchers have identified particular sequences and signals in
RNAs that have the potential for having a specific effect on RNA
stability. In certain embodiments of the invention, therefore,
there is a desire to optimize expression of the disclosed nucleic
acid segments in planta. One particular method of doing so, is by
alternation of the bacterial gene to remove sequences or motifs
which decrease expression in a transformed plant cell. The process
of engineering a coding sequence for optimal expression in plants
is often referred to as "plantizing" a DNA sequence.
Particularly problematic sequences are those which are A+T rich.
Unfortunately, since B. thuringiensis has an A+T rich genome,
native crystal protein gene sequences must often be modified for
optimal expression in a plant. The sequence motif ATTA (or AUUUA as
it appears in RNA) has been implicated as a destabilizing sequence
in mammalian cell mRNA (Shaw and Kamen, 1986). Many short lived
mRNAs have A+T rich 3' untranslated regions, and these regions
often have the ATTA sequence, sometimes present in multiple copies
as multimedia (e.g., ATTTATTTA . . . ). Shaw and Kamen showed that
the transfer of the 3' end of an unstable mRNA to a stable RNA
(globin or VA1) decreased the stable RNA's half life dramatically.
They further showed that a pentamer of ATTTA had a profound
destabilizing effect on a stable message, and that this signal
could exert its effect whether it was located at the 3' end or
within the coding sequence. However, the number of ATTTA sequences
and/or the sequence context in which they occur also appear to be
important in determining whether they function as destabilizing
sequences. Shaw and Kamen showed that a trimer of ATTTA had much
less effect than a pentamer on mRNA stability and a dimer or a
monomer had no effect on stability (Shaw and Kamen, 1987). Note
that multimers of ATTA such as a pentamer automatically create an
A+T rich region. This was shown to be a cytoplasmic effect, not
nuclear. In other unstable mRNAs, the ATTTA sequence may be present
in only a single copy, but it is often contained in an A+T rich
region. From the animal cell data collected to the date, it appears
that ATTTA at least in some contexts is important in stability, but
it is not yet possible to predict with occurrences of ATTTTA are
destabiling elements or whether any of these effects are likely to
be seen in plants.
Some studies on mRNA degradation in animal cells also indicate that
RNA degradation may begin in some cases with nucleolytic attack in
A+T rich regions. It is not clear if these cleavages occur at ATTTA
sequences. There are also examples of mRNAs that have differential
stability depending on the cell type in which they are expressed or
on the stage within the cell cycle at which they are expressed. For
example, histone mRNAs are stable during DNA synthesis but unstable
if DNA synthesis is disrupted. The 3' end of some histidine mRNAs
seems to be responsible for this effect (Pandey and Marzluff,
1987). It does not appear to be mediated by ATTTA, nor it is clear
what controls the differential stability of this mRNA. Another
example is the differential stability of IgG mRNA in B lymphocytes
during B cell maturation (Genovese and Milcarek, 1998). A final
example is the instability of a mutant .beta.-thallesemic globin
mRNA. In bone marrow cells, where this gene is normally expressed,
the mutant mRNA is unstable, while the wild-type mRNA is stable.
When the mutant gene is expressed in HeLa or L cells in vitro, the
mutant mRNA shows no instability (Lim et al., 1988). These examples
all provide evidence that mRNA stability can be mediated by cell
type or cell cycle specific factors. Furthermore this type of
instability is not yet associated with specific sequences. Given
these uncertainities, it is not possible to predict which RNAS are
likely to be unstable in a given cell. In addition, even the ATTTA
motif may act differentially depending on the nature of the cell in
which the RNA is present. Shaw and Kamen (1987) have reported that
activation of protein kinase C can block degradation mediated by
ATTTA.
The addition of a polyadenylation string to the 3' end is common to
most eukaryotic mRNAs, both plant and animal. The currently
accepted view of polyA addition is that the nascent transcript
extends beyond the mature 3' terminus. Contained within this
transcript are signals for polyadenylation and proper 3' end
formation. This processing at the 3' end involves cleavage of the
mRNA and addition of the polyA to the mature 3' end. By searching
for consensus sequences near the polyA tract in both plant and
animal mRNAs, it has been possible to identify consensus sequences
that apparently are involved in polyA addition and 3' end cleavage.
The same consensus sequences seem to be important to both of these
processes. These signals are typically a variation on the sequence
AATAAA. In animal cells, some variants of this sequence that are
functional have been identified; in plant cells there seems to be
an extended range of functional sequences (Wickens and Stephenson,
1984; Dean et al., 1986). Because all of these consensus sequences
are variations on AATAAA, they all are A+T rich sequences. This
sequence is typically found 15 to 20 bp before the polyA tract is a
mature mRNA. Studies in animal cells indicate that this sequence is
involved in both polyA addition and 3' maturation. Site directed
mutations in this sequence can disrupt these functions (Conway and
Wickens, 1988; Wickens et al., 1987). However, it has also been
observed that sequences up to 50 to 100 bp 3' to the putative polyA
signal are also required, i.e., a gene that has a normal AATAAA but
has been replaced or disrupted downstream does not gel properly
polyadenylated (Gil and Proudfoot, 1984; Sadofsky and Alwine, 1984;
McDevitt et al., 1984). That is, the polyA signal itself is not
sufficient for complete and proper processing. It is not yet known
what specific downstream sequences are required in addition to the
polyA signal, or if there is a specific sequence that has time
function. Therefore, sequence analysis can only identify potential
polyA signals.
In naturally occurring mRNAs that are normally polyadenylated, it
has been observed that disruption of this process, either by
altering the polyA signal or other sequences in the mRNA, profound
effects can be obtained in the level of functional mRNA. This has
been observed in several naturally occurring mRNAs, with results
that are gene-specific so far.
It has been shown that in natural mRNAs proper polyadenylation is
important in mRNA accumulation, and that disruption of this process
can effect mRNA levels significantly. However, insufficient
knowledge exists to predict the effect of changes in a normal gene.
In a heterologous gene, it is even harder to predict the
consequences. However, it is possible that the putative sites
identified are dysfunctional. That is, these sites may not act as
proper polyA sites, but instead function as aberrant sites that
give rise to unstable mRNAs.
In animal cell systems, AATAAA is by far the most communication
signal identified in mRNAs upstream of the polyA, but at least four
variants have also been found (Wickens and Stephenson, 1984). In
plants, not nearly so much analysis has been done, but it is clear
that multiple sequences similar to AATAAA can be used. The plant
sites in Table 5 called major or minor refer only to the study of
Dean et al. (1986) which analyzed only three types of plant gene.
The designation of polyadenylation sites as major or minor refers
only to the frequency of their occurrence as functional sites in
naturally occurring genes that have been analyzed. In the case of
plants this is a very limited database. It is hard to predict with
any certainty that a site designated major or minor is more or less
likely to function partially or completely when found in a
heterologous gene such as those encoding the crystal proteins of
the present invention.
TABLE-US-00005 TABLE 5 POLYADENYLATION SITES IN PLANT GENES PA
AATAAA Major consensus site P1A AATAAT Major plant site P2A AACCAA
Minor plant site P3A ATATAA '' P4A AATCAA '' P5A ATACTA '' P6A
ATAAAA '' P7A ATGAAA '' P8A AAGCAT '' P9A ATTAAT '' P10A ATACAT ''
P11A AAAATA '' P12A ATTAAA Minor animal site P13A AATTAA '' P14A
AATACA '' P15A CATAAA ''
The present invention provides a method for preparing synthetic
plant genes which genes express their protein product at levels
significantly higher than the wild-type gene which were commonly
employed in plant transformation heretofore. In another aspect, the
present invention also provides novel synthetic plant genes which
encode non-polar proteins.
As described above, the expression of native B. thuringiensis genes
in plants is often problematic. The nature of the coding sequences
of B. thuringiensis genes distinguishes them from plant genes as
well as many other heterologous genes expressed in plants. In
particular, B. thuringiensis genes are very rich (-62%) in adenine
(A) and thymine (T) while plant genes and most other bacterial
genes which have been expressed in plants are on the order of
45-55% A+T.
Due to the degeneracy of the genetic code and the limited number of
codon choices for any amino acid, most of the "excess" A+T of the
structural coding sequences of some Bacillus species are found in
the third position of the codons. That is, gene of some Bacillus
species have A or T as the third nucleotide in many codons. Thus
A+T content in part can determine codon usage bias. In addition, it
is clear that genes evolve for maximum function in the organism in
which they evolve. This means that particular nucleotide sequences
found in a gene from one organism, where they may play no role
except to code for a particular stretch of amino acids, have the
potential to be recognized as gene control elements in another
organism (such as transcriptional promoters or terminators, polyA
addition sites, intron splice sites, or specific mRNA degradation
signals). It is perhaps surprising that such misread signals are
not a more common feature of heterologous gene expression, but this
can be explained in part by the relatively homogeneous A+T content
(.about.50%) of many organisms. This A+T content plus the nature of
the genetic code put clear constraints on the likelihood of
occurrence of any particular oligonucleotide sequence. Thus, a gene
from E. coli with a 50% A+T content is much less likely to contain
nay particular A+T rich segment then a gene from B.
thuringiensis.
Typically, to obtain high-level expression of the S-endotoxin genes
in plants, existing structural coding sequences ("structural gene")
which codes for the S-endotoxin are modified by removal of ATTTA
sequences and putative polyadenylation signals by site directed
mutagenesis of the DNA comprising the structural gene. It is most
preferred that substantially all the polyadenylation signals and
ATTTA sequences are removed although enhanced expression levels are
observed with only partial removal of either of the above
identified sequences. Alternately if a synthetic gene is prepared
which codes for the expression of the subject protein, codons are
selected to avoid the ATTTA sequence and putative polyadenylation
signals. For purposes of the present invention putative
polyadenylation signals include, but are not necessarily limited
to, AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA,
AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA AATACA and CATAAA.
In replacing the ATTA sequences and polyadenylation signals, codons
are preferably utilized which avoid the codons which are rarely
found in plant genomes.
The selected DNA sequence is scanned to identify regions with
greater than four consecutive adenine (A) or thymine (T)
nucleotides. The A+T regions are scanned for potential plant
polyadenylation signals. Although the absence of five or more
consecutive A or T nucleotides eliminates most plant
polyadenylation signals, if there are more than one of the minor
polyadenylation signals identified within ten nucleotides of each
other, then the nucleotide sequence of this region is preferably
altered to remove these signals while maintaining the original
encoded amino acid sequence.
The second step is to consider the about 15 to about 30 or so
nucleotide residues surrounding the A+T rich region identified in
step one. If the A+T content of the surrounding region is less than
80%, the region should be examined for polyadenylation signals.
Alteration of the region based on polyadenylation signals is
dependent upon (1) the number of polyadenylation signal present and
(2) presence of a major plant polyadenylation signal.
The extended region is examined for the presence of plant
polyadenylation signals. The polyadenylation signals are removed by
site-directed mutagenesis of the DNA sequence. The extended region
is also examined for multiple copies of the ATTTA sequence which
are also removed by mutagenesis.
It is also preferred that regions comprising many consecutive A+T
bases or G+C bases are disrupted since these regions are predicted
to have a higher likelihood to form hairpin structure due to
self-complementarity. Therefore, insertion of heterogeneous base
pairs would reduce the likelihood of self-complementary secondary
structure formation which are known to inhibit transcription and/or
translation in some organisms. In most cases, the adverse effects
may be minimized by using sequences which do not contain more than
five consecutive A+T or G+C.
4.11.5 Synthetic Oligonucleotides for Mutagenesis
When oligonucleotides are used in the mutagenesis, it is desirable
to maintain the proper amino acid sequence and reading frame,
without introducing common restriction sites such as GglII,
HindIII, SacI, KpnI, EcoRI, NcoI, PstI and SalI into the modified
gene. These restriction sites are found in poly-linker insertion
sites of many cloning vectors. Of course, the introduction of new
polyadenylation signals, ATTTA sequences or consecutive stretches
of more than five A+T or G+C, should also be avoided. The preferred
size for the oligonucelotides is about 40 to about 50 bases, but
fragments ranging from about 18 to about 100 bases have been
utilized. In most cases, a minimum of about 5 to about 8 base pairs
of homology to the template DNA on both ends of the synthesized
fragment are maintained to insure proper hybridization of the
primer to the template. The oligonucleotides should avoid sequences
longer than five base pairs A+T or G+C. Codons used in the
replacement of wild-type codons should preferably avoid the TA or
CG doublet wherever possible. Codons are selected from a plant
preferred codon table (such as Table 6 below) so as to avoid codons
which are rarely found in plant genomes, and efforts should be made
to select codons to preferably adjust the G+C content to about
50%.
TABLE-US-00006 TABLE 6 PREFERRED CODON USAGE IN PLANTS Percent
Usage Amino Acid Codon in Plants ARG CGA 7 CGC 11 CGG 5 CGU 25 AGA
29 AGG 23 LEU CUA 8 CUC 20 CUG 10 CUU 28 UUA 5 UUG 30 SER UCA 14
UCC 26 UCG 3 UCU 21 AGC 21 AGU 15 THR ACA 21 ACC 41 ACG 7 ACU 31
PRO CCA 45 CCC 18 CCG 9 CCU 26 ALA GCA 23 GCC 32 GCG 3 GCU 41 GLY
GGA 32 GGC 20 GGG 11 GGU 37 ILE AUA 12 AUC 45 AUU 43 VAL GUA 9 GUC
20 GUG 28 GUU 43 LYS AAA 36 AAG 64 ASN AAC 72 AAU 28 GLN CAA 64 CAG
36 HIS CAC 65 CAU 35 GLU GAA 48 GAG 52 ASP GAC 48 GAU 52 TYR UAC 68
UAU 32 CYS UGC 78 UGU 22 PHE UUC 56 UUU 44 MET AUG 100 TRP UGG
100
Regions with many consecutive A+T bases or G+C bases are predicted
to have a higher likelihood to form hairpin structures due to
self-complementary. Disruption of these regions by the insertion of
heterogeneous base pairs is preferred and should reduce the
likelihood of the formation of self-complementary secondary
structures such as hairpins which are known in some organisms to
inhibit transcription (transcriptional terminators) and translation
(attenuators).
Alternatively, a completely synthetic gene for a given amino acid
sequence can be prepared, with regions of five or more consecutive
A+T or G+C nucleotides being avoided. Codons are selected avoiding
the TA and CG doublets in codons whenever possible. Codon usage can
be normalized against a plant preferred codon usage table (such as
Table 6) and the G+C content preferably adjusted to about 50%. The
resulting sequence should be examined to ensure that there are
minimal putative plant polyadenylation signals and ATTTA sequences.
Restriction sites found in commonly used cloning vectors are also
preferably avoided. However, placement of several unique
restriction sites throughout the gene is useful for analysis of
gene expression or construction of gene variants.
4.11.6 "Plantized" Gene Constructs
The expression of a plant gene which exists in double-stranded DNA
form involves transcription of messenger RNA (mRNA) from one strand
of the DNA by RNA polymerase enzyme, and the subsequent processing
of the mRNA primary transcript inside the nucleus. This processing
involves a 3' non-translated region which adds polyadenylate
nucleotides to the 3' end of the RNA. Transcription of DNA into
mRNA is regulated by a region of DNA usually referred to as the
"promoter." The promoter region contains a sequence of bases that
signals RNA polymerase to associate with the DNA and to initiate
the transcription of mRNA using one of the DNA strands as a
template to make a corresponding strand of RNA.
A number of promoters which are active in plant cells have been
described in the literature. These include the nopaline synthase
(NOS) and octopine synthase (OCS) promoters (which are carried on
tumor-inducing plasmids of Agrobacerium tumefaciens), the
Cauliflower Mosaic Virus (CaMV) 19S and 35S promoters, the
light-inducible promoter from the small subunit of ribulose
bis-phosphate carboxylase (ssRUBISCO, a very abundant plant
polypeptide) and the mannopine synthase (MAS) promoter (Velten et
al., 1984 and Velten and Schnell, 1985). All of these promoters
have bene used to create various types of DNA constructs which have
been expressed in plants (see e.g., Int. Pat. Appl. Publ. No. WO
84/02913).
Promoters which are known or are found to cause transcription of
RNA in plant cells can be used in the present invention. Such
promoters maybe obtained from plants or plant viruses and include,
but are not limited to, the CaMV35S promoter and promotes isolated
from plant genes such as ssRUBISCO genes. As described below, it is
preferred that the particular promoter selected should be capable
of causing sufficient expression to result in the production of an
effective amount of protein.
The promoters used in the DNA constructs (i.e. chimeric plant
genes) of the present invention may be modified, if desired, to
affect their control characteristics. For example, the CaMV35S
promoter may be ligated to the portion of the ssRUBISCO gene that
represents the expression of SSRUBISCO in the absence of light, to
create a promoter which is active in leaves but not in roots. The
resulting chimeric promoter may be used as described herein. For
purposes of this description, the phrase "CaMV35S" promoter thus
includes variations of CaMV35S promoter, e.g., promoters derived by
means of ligation with operator regions, random or controlled
mutagenesis, etc. Furthermore, the promoters may be altered to
contain multiple "enhancer sequences" to assist in elevating gene
expression.
The RNA produced by a DNA construct of the present invention also
contains a 5' non-translated leader sequence. This sequence can be
derived from the promoter selected to express the gene, and can be
specifically modified so as to increase translation of the mRNA.
The 5' non-translated regions can also be obtained from viral
RNA's, from suitable eukaryotic genes, or from a synthetic gene
sequence. The present invention is not limited to constructs, as
presented in the following examples. Rather, the non-translated
leader sequence can be part of the 5' end of the non-translated
region of the coding sequence for the virus coat protein, or part
of the promoter sequence, or can be derived from an unrelated
promoter or coding sequence. In any case, it is preferred that the
sequence flanking the initial site conform to the translational
consensus sequence rules for enhanced translation initiation
reported by Kozak (1984).
The cry DNA constructs of the present invention may also contain
one or more modified or fully-synthetic structural condign
sequences which have been changed to enhance the performance of the
cry gene in plants. The structural genes of the present invention
may optionally encode a fusion protein comprising an amino-terminal
chloroplast transit peptide or secretory signal sequence.
The DNA construct also contains a 3' non-translated region. The 3'
non-translated region contains a polyadenylation signal which
functions in plants to cause the addition of polyadenylate
nucleotides to the 3' end of the viral RNA. Example of suitable 3'
regions are (1) the 3' transcribed, non-translated regions
containing the polyadenylation signal of Agrobacterium
tumor-inducing (Ti) plasmid genes, such as the nopaline synthase
(NOS) gene, and (2) plant genes like the soybean storage protein
(7S) genes and the small subunit of the RuBP carboxylase (E9)
gene.
4.12. Methods for Producing Insect-resistant Transgenic Plants
By transforming a suitable host cell, such as a plant cell, with a
recombinant cry* gene-containing segment, the expression of the
encoded crystal protein (i.e., a bacterial crystal protein or
polypeptide having insecticidal activity against coleopterans) can
result in the formation of insect-resistant plants.
By way of example, one may utilize an expression vector containing
a coding region for a B. thuringiensis crystal protein and an
appropriate selectable marker to transform a suspension of
embryonic plant cells, such as wheat or corn cells using a method
such as particle bombardment (Maddock et al., 1991; Vasil et al.,
1992) to deliver the DNA coated on microprojectiles into the
recipient cells. Transgenic plants are then regenerated from
transformed embryonic calli that expresses the insecticidal
proteins.
The formation of transgenic plants may also be accomplished using
other methods of cell transformation which are known in the art
such as Agrobacterium-mediated DNA transfer (Fraley et al., 1983).
Alternatively, DNA can be introduced into plants by direct DNA
transfer into pollen (Zhou et al., 1983; Hess, 1987; Luo et al.,
1998), by injection of the DNA into reproductive organs of a plant
(Pena et al., 1987), or by direct injection of DNA into the cells
of immature embryos followed by the rehydration of desicated
embryos (Neuhaus et al., 1987; Benbrook et al. 1986).
The regeneration, development, and cultivation of plants from
single plant protoplast transformants or from various transformed
explants is well known in the art (Weissbach and Weissbach, 1988).
This regeneration and growth process typically includes the steps
of selection of transformed cells, culturing those individualized
cells through the usual stages of embryonic development through the
rooted plantlet stage. Transgenic embryos and seeds are similarly
regenerated. The resulting transgenic rooted shoots are thereafter
planted in an appropriate plant growth medium such as soil.
The development or regeneration of plants containing the foreign,
exogenous gene that encodes a polypeptide of interest introduced by
Agrobacterium from leaf explants can be achieved by methods well
known in the art such as described (Horsch et al., 1985). In this
procedure, tranformants are cultured in the presence of a selection
agent and in a medium that induces the regeneration of shoots in
the plant string being transformed as described (Fraley et al.,
1983).
This procedure typically produces shoots within two to four months
and those shoots are then transferred to an appropriate
root-inducing medium containing the selective agent and a
antibiotic to prevent bacterial growth. Shoots that rooted in the
presence of the selective agent to form plantlets are then
transplanted to soil or other media to allow the production of
roots. These procedures vary depending upon the particular plant
strain employed, such variations being well known in the art.
Preferably, the regenerated plant are self-pollinated to provide
homozygous transgenic plants, as discussed before. Otherwise,
pollen obtained from the regenerated plant is crossed to seed-grown
plants of agronomically important, preferably inbred lines.
Conversely, pollen from plants of those important lines is used to
pollinate regenerated plants. A transgenic plant of the present
invention containing a desired polypeptide is cultivated using
methods well known to one skilled in the art.
Such plants can form germ cells and transmit the transformed
trait(s) to progeny plants. Likewise, transgenic plants can be
grown in the normal manner and crossed with plants that have the
same transformed hereditary factors or other hereditary factors.
The resulting hybrid individuals have the corresponding phenotypic
properties. A transgenic plant of this invention thus has an
increased amount of a coding region (e.g, a mutated cry gene) that
encodes the mutated Cry polypeptide of interest. A preferred
transgenic plant is an independent segregant and can transmit that
gene and its activity to its progeny. A more preferred transgenic
plant is homozygous for that gene, and transmits that gene to all
of its offspring on sexual mating.
Seed from a transgenic plant may be grown in the field or
greenhouse, and resulting sexually mature transgenic plants are
self-pollinated to generate true breeding plants. The progeny from
these plants become true breeding lines that are evaluated for, by
way of example, increased insecticidal capacity against coleopteran
insects, preferably in the field, under a range of environment
conditions. The inventors contemplate that the present invention
will find particular utility in the creation of transgenic plants
of commercial interest including various grasses, grains, fibers,
tubers, legumens, ornamental plants, cacti, succulents, fruits,
berries, and vegetables, as well as a number of nut- and
fruit-bearing trees and plants.
4.13 Methods for Producing Combinatorial Cry3* Variants
Crystal protein mutants containing substitutions in one or more
domains may be constructed via a number of techniques. For
instance, sequences of highly related genes can be readily shuffled
using the PCR.TM.-based technique described by Stemmer (1994).
Alternatively, if suitable restriction sites are available, the
mutations of one cry gene may be combined with the mutations of a
second cry gene by routine subcloning methodologies. If a suitable
restriction site is not available, one may be generated by
oligonucleotide directed mutagenesis using any number of procedures
known to those skilled in the art. Alternatively, splice-overlap
extension PCR.TM. (Horton et al., 1989) may be used to combine
mutations in different regions of a crystal protein. In this
process, overlapping DNA fragments generated by the PCR.TM. and
containing different mutations within their unique sequences may be
annealed and used as a template for amplification using flanking
primers to generate a hybrid gene sequence. Finally, cry* mutants
may be combined by simply using one cry mutant as a template for
oligonucleotide-directed mutagenesis using any number of protocols
such as those described herein.
4.14 Isolating Homologous Gene and Gene Fragments
The genes and .delta.-endotoxins according to the subject invention
include not only the full length sequences disclosed herein but
also fragments of these sequences, or fusion proteins, which retain
the characteristics insecticidal activity of the sequences
specifically exemplified herein.
It should be apparent to a person skilled in the art that
insecticidal .delta.-endotoxins can be identified and obtained
through several means. The specific genes, or portions thereof, may
be obtained from a culture depository, or constructed
synthetically, for example, by use of a gene machine. Variations of
these genes may be readily constructed using standard techniques
for making point mutations. Also, fragments of these genes can be
made using commercially available exonucleases or endonucleases
according to standard procedures. For example, enzymes such as
Bal31 or site-directed mutagenesis can be used to systematically
cut off nucleotides from the ends of these genes. Also, genes which
code for active fragments may be obtained using a variety of other
restriction enzymes. Proteases may be used to directly obtain
active fragments of these .delta.-endotoxins.
Equivalent .delta.-endotoxins and/or genes encoding these
equivalent .delta.-endotoxins can also be isolated from Bacillus
strains and/or DNA libraries using the teachings provided herein.
For example, antibodies to the .delta.-endotoxins disclosed and
claimed herein can be used to identify and isolate other
.delta.-endotoxins from a mixture of proteins. Specifically,
antibodies may be raised to portions of the .delta.-endotoxins
which are most constant and most distinct from other B.
thuringiensis .delta.-endotoxins. These antibodies can then be used
to specifically identify equivalent .delta.-endotoxins with the
characteristic insecticidal activity by immunoprecipitation, enzyme
linked immunoassay (ELISA), or Western blotting.
A further method for identifying the .delta.-endotoxins and genes
of the subject invention is through the use of oligonucleotide
probes. These probes are nucleotide sequences having a detectable
label. As is well known in the art, if the probe molecule and
nucleic acid sample hybridize by forming a strong bond between the
two molecules, it can be reasonably assumed that the probe and
sample are essentially identical. The probe's detectable label
provides a means for determining in a known manner whether
hybridization has occurred. Such a probe analysis provides a rapid
method for identifying fomicidal .beta.-endotoxin genes of the
subject invention.
The nucleotide segments which are used as probes according to the
invention can be synthesized by use of DNA synthesizers using
standard procedures. In the use of the nucleotide segments as
probes, the particular probe is labeled with any suitable label
known to those skilled in the art, including radioactive and
non-radioactive labels. Typical radioactive labels include
.sup.32p, .sup.125I, .sup.35S, or the like. A probe labeled with a
radioactive isotope can be constructed from a nucleotide sequence
complementary to the DNA sample by a conventional nick translation
reaction, using a DNAase and DNA polymerase. The probe and sample
can then be combined in a hybridization buffer solution and held at
an appropriate temperature until annealing occurs. Thereafter, the
membrane is washed free of extraneous materials, leaving the sample
and bound probe molecules typically detected and quantified by
autoradiography and/or liquid scintillation counting.
Non-radioactive labels include, for example, ligands such as biotin
or thyroxine, as well as enzymes such as hydrolases or peroxidases,
or the various chemiluminescers such as luciferin, or fluorescent
compounds like fluorescein and its derivatives. The probe may also
be labeled at both ends with different types of labels for ease of
separation as, for example, by using an isotopic label at the end
mentioned above and a biotin label at the other end.
Duplex formation and stability depend on substantial
complementarity between the two strands of a hybrid, and, as noted
above, a certain degree of mismatch can be tolerated. Therefore,
the probes of the subject invention include mutations (both single
and multiple), deletions, insertions of the described sequences,
and combinations thereof, wherein said mutations, insertions and
deletions permit formation of stable hybrids with the target
polynucleotide of interest. Mutations, insertions, and deletions
can be produced in a given polynucleotide sequence in many ways, by
methods currently known to an ordinarily skilled artisan, and
perhaps by other methods which may become known in the future.
The potential variations in the probes listed is due, in part, to
the redundancy of the genetic code, i.e., more than one coding
nucleotide triple (codon) can be used for most of the amino acids
used to make proteins. Therefore difficult nucleotide sequences can
code for a particular amino acid. Thus, the amino acid sequences of
the B. thuringiensis .delta.-endotoxins and peptides can be
prepared by equivalent nucleotide sequences encoding the same amino
acid sequence of the protein or peptide. Accordingly, the subject
invention includes such equivalent nucleotide sequences. Also,
inverse or completed sequences are an aspect of the subject
invention and can be readily used by a person skilled in this art.
In addition, it has been shown that proteins of identified
structure and function may be constructed by changing the amino
acid sequence if such changes do not alter the protein secondary
structure (Kaiser and Kezdy, 1984). Thus, the subject invention
includes mutants of the amino acid sequence depicted herein which
do not alter the protein secondary structure, or if the structure
is altered, the biological activity is substantially retained.
Further, the invention also includes mutants of organisms hosting
all or part of a .delta.-endotoxin encoding a gene of the
invention. Such mutants can be made by techniques well known to
persons skilled in the art. For example, UV irradiation can be used
to prepare mutants of host organisms. Likewise, such mutants may
include asprogenous host cells which also can be prepared by
procedures well known in the art.
4.15 Ribozymes
Ribozymes are enzymatic RNA molecules which cleave particular mRNA
species. In certain embodiments, the inventors contemplate the
selection and utilization of ribozymes capable of cleaving the RNA
segments of the present invention, and their use to reduce activity
of target mRNAs in particular cell types or tissues.
Six basic varieties of naturally-occurring enzymatic RNAs are known
presently. Each can catalyze the hydrolysis of RNA phosphodiester
bonds in trans (and thus can cleave other RNA molecules) under
physiological conditions. In general, enzymatic nucleic acids act
by first binding to a target RNA. Such binding occurs through the
target binding portion of a enzymatic nucleic acid which is held in
close proximity to an enzymatic portion of the molecule that acts
to cleave the target RNA. Thus, the enzymatic nucleic acid first
recognizes and then binds a target RNA through complementary
base-pairing, and once bound to the correct site, acts
enzymatically to cut the target RNA. Strategic cleavage of such a
target RNA will destroy its ability to direct synthesis of an
encoded protein. After an enzymatic nucleic acid has bound and
cleaved in RNA target, it is released from that RNA to search for
another target and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is advantageous over many
technologies, such as antisense technology (where a nucleic acid
molecule simply binds to a nucleic acid target to block its
translation) since the concentration of ribozyme necessary to
affect a therapeutic treatment is lower than the of an antisense
oligonucleotide. This advantage reflects the ability of the
ribozyme to act enzymatically. Thus, a single ribozyme molecule is
able to cleave many molecules of target RNA. In addition, the
ribozyme is a highly specific inhibitor, with the specificity of
inhibition depending not only on the base pairing mechanism of
binding to the target RNA, but also on the mechanism of target RNA
cleavage. Single mismatches, or base-substitutions, near the site
of cleavage can completely eliminate catalytic activity of a
ribozyme. Similar mismatches in antisense molecules do not prevent
their action (Woolf et al., 1992). Thus, the specificity of action
of a ribozyme is greater than that of antisense oligonucleotide
binding the same RNA site.
The enzymatic nucleic acid molecule may be formed in a hammerhead,
hairpin, a hepatitis .delta. virus, group I intron or RNase PRNA
(in association with an RNA guide sequence) or Neurospora VS RNA
motif. Examples of hammerhead motifs are described by Rossi et al.
(1992); examples of hairpin motifs are described by Hampel et al.
(Eur. Pat. EP 0360257), Harnpel and Triz (1989), Hampel et al.
(1990) and Cech et al. (U.S. Pat. No. 5,631,359; an example of the
hepatitis .delta. virus motif is described by Perrotta and Been
(1992); an example of the RNaseP motif is described by
Guerrier-Takada et al. (1983); Neurospora VS RNA ribozyme motif is
described by Collins (Saville and Collins, 1990; Saville and
Collins, 1991; Collins and Olive, 1993); and an example of the
Group 1 intron is described by Cech et al. (U.S. Pat. No.
4,987,071). All that is important in an enzymatic nucleic acid
molecule of this invention is that it has a specific substrate
binding site which is complementary to one or more of the target
gene RNA regions, and that it have nucleotide sequences within or
surrounding that substrate binding site which impart an RNA
cleaving activity to the molecule. Thus the ribozyme constructs
need not be limited to specific motifs mentioned herein.
The invention provides a method for producing a class of enzymatic
cleaving agents which exhibit a high degree of specificity for the
RNA of a desired target. The enzymatic nucleic acid molecule is
preferably targeted to a highly conserved sequence region of a
target mRNA such that specific treatment of a disease or condition
can be provided with either one or several enzymatic nucleic acids.
Such enzymatic nucleic acid molecules can be delivered exogenously
to specific cells as required. Alternatively, the ribozymes can be
expressed from DNA or RNA vectors that are delivered to specific
cells.
Small enzymatic nucleic acid motifs (e.g., of the hammerhead or the
hairpin structure) may be used for exogenous delivery. The simple
structure of these molecules increases the ability of the enzymatic
nucleic acid to invade targeted regions of the mRNA structure.
Alternatively, catalytic RNA molecules can be expressed within
cells from eukaryotic promoters (e.g., Scanlon et al., 1991;
Kashani-Sabet et al., 1992; Dropulic et al., 1992; Weerasinghe et
al., 1991; Ojwang et al., 1992; Chen et al., 1992; Sarver et al.,
1990). Those skilled in the art realize that any ribozyme can be
expressed in eukaryotic cells from the appropriate DNA vector. The
activity of such ribozymes can be augmented by their release from
the primary transcript by a second ribozyme (Draper et al., Int.
Pat. Appl. Publ. No. WO 93/23569, and Sullivan et al., Int. Pat.
Appl. Publ. No. WO 94/02595, both hereby incorporated in their
totality by reference herein; Ohkawa et al., 1992; Taira et al.,
1991; Ventura et al., 1993).
Ribozymes may be added directly, or can be completed with cationic
lipids, lipid complexes, packaged within liposomes, or otherwise
delivered to target cells. The RNA or RNA complexes can be locally
administered to relevant tissues ex vivo, or in vivo through
injection, aerosol inhalation, infusion pump or stent, with or
without their incorporation in biopolymers.
ribozymes may be designed as described in Draper et al. (Int. Pat.
Appl. Publ. No. WO 93/23569), or Sullivan et al., (Int. Pat. Appl.
Publ. No. WO 94/02595) and synthesized to be tested in vitro and in
vitro, as described. Such ribozymes can also be optimized for
delivery. While specific examples are provided, those in the art
will recognize that equivalent RNA targets in other species can be
utilized when necessary.
Hammerhead or hairpin ribozymes may be individually analyzed by
computer folding (Jaeger et la., 1989) to assess whether the
ribozyme sequences fold into the appropriate secondary structure.
Those ribozymes with unfavorable intramolecular interactions
between the binding arms and the catalytic core are eliminated from
consideration. Varying binding arm lengths can be chosen to
optimize activity. Generally, at least 5 bases on each arm are able
to bind to, or otherwise interact with, the target RNA.
Ribozymes of the hammerhead or hairpin motif may be designed to
anneal to various sites in the mRNA message, and can be chemically
synthesized. The method of synthesis used follows the procedure for
normal RNA synthesis as described in Usman et al. (1987) and in
Scaringe et al. (1990) and makes use of common nucleic acid
protecting and coupling groups, such as dimethoxytrityl at the
5'-end, and phosboramidites at the 3'-end. Average stepwise
coupling yields are typically>98%. Hairpin ribozymes may be
synthesized in two parts and annealed to reconstruct an active
ribozyme (Chowrira and Burke, 1992). Ribozymes may be modified
extensively to enhance stability by modification with nuclease
resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro,
2'-o-methyl, 2'-H (for a review see Usman and Cedergren, 1992).
Ribozymes may be purified by gel electrophoresis using general
methods or by high pressure liquid chromatography and resuspended
in water.
Ribozyme activity can be optimized by altering the length of the
ribozyme binding arms, or chemically synthesizing ribozymes with
modifications that prevent their degradation by serum ribonucleases
(see e.g. Int. Pat. Appl. Publ. No. WO 92/07065; Perrault et al,
1990; Pieken et al., 1991; Usman and Cedergren, 1992; Int. Pat.
Appl. Publ. No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162;
Eur. Pat. Appl. Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and
Int. Pat. Appl. Publ. No. WO 94/13688, which describe various
chemical modifications that can be made to the sugar moieties of
enzymatic RNA molecules), modifications which enhance their
efficacy in cells, and removal of stem II bases to shorten RNA
synthesis times and reduce chemical requirements.
Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes
the general methods for delivery of enzymatic RNA molecules.
Ribozymes may be administered to cells by a variety of methods
known to those familiar to the art, including, but not restricted
to, encapsulation in liposomes, by iontophoresis, or by
incorporation into other vehicles, such as hydrogels,
cyclodextrins, biodegradable nanocapsules, and bioadhesive
microspheres. For some indications, ribozymes may be directly
delivered ex vivo to cells or tissues with or without the
aforementioned vehicles. Alternatively, the RNA/vehicle combination
may be locally delivered by direct inhalation, by direct injection
or by use of a catheter, infusion pump or stent. Other routes of
delivery include, but are not limited to, intravascular,
intramuscular, subcutaneous or joint injection, aerosol inhalation,
oral (tablet or pill form), topical, systemic, ocular,
intraperitoneal and/or intrathecal delivery. More detailed
descriptions of ribozyme delivery and administration are provided
in Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) and
Draper et al. (Int. Pat. Appl. Publ. No. WO 93/23659) which have
been incorporated by reference herein.
Another means of accumulating high concentrations of a ribozyme(s)
within cells is to incorporate the ribozyme-encoding sequences into
a DNA expression vector. Transcription of the ribozyme sequences
are driven from a promoter for eukaryotic RNA polymerasse I (pol
I), RNA polymerase II (pol II), or RNA polymerase III (pol III).
Transcripts from pol II or pol III promoters will be expressed at
high levels in all cells; the levels of a given pol II promoter in
a given cell type will depend on the nature of the gene regulatory
sequences (enhancers, silencers, etc.) present nearby. Prokaryotic
RNA polymerase promoters may also be used, providing that the
prokaryotic RNA polymerase enzyme is expressed in the appropriate
cells (Elroy-Stein and Moss, 1990; Gao and Huang, 1993; Lieber et
al., 1993; Zhou et al.; 1990). Ribozymes expressed from such
promoters can function in mammalian cells (e.g. Kashani-Saber et
al., 1992; Ojwang et al., 1992; Chen et al., 1992; Yu et al., 1993;
L'Huillier et al., 1992; Lisziewicz et al., 1993). Such
transcription units can be incorporated into a variety of vectors
for introduction into mammalian cells, including but not restricted
to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or
adeno-associated vectors), or viral RNA vectors (such as
retroviral, semliki forest virus, sindbis virus vectors).
Ribozymes of this invention may be used as diagnostic tools to
examine genetic drift and mutations within cell lines or cell
types. They can also be used to assess levels of the target RNA
molecule. The close relationship between ribozyme activity and the
structure of the target RNA allows the detection of mutations in
any region of the molecule which alters the base-pairing and
three-dimensional structure of the target RNA. By using multiple
ribozymes described in this invention, one may map nucleotide
changes which are important to RNA structure and function in vitro,
as well as in cells and tissues. Cleavage of target RNAs with
ribozymes may be used to inhibit gene expression and define the
role (essentially) of specified gene products in particular cells
or cell types.
5.0 EXAMPLES
The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
5.1 Example 1
Three-dimensional Structure Cry3Bb
The three-dimensional structure of Cry3Bb was determined by X-ray
crystallography. Crystallization of Cry3Bb and X-ray diffraction
data collection were performed as described by Cody et al. (1992).
The crystal structure of Cry3Bb was refined to a residual R factor
of 18.0% using data collected to 2.4 .ANG. resolution. The crystals
belong to the space group C222, with unit cell dimensions a=122.44,
b=131.81, and c=105.37 .ANG. and contain one molecule in the
asymmetric unit. Atomic coordinates for Cry3Bb are described in
Example 31 and listed in Section 9.
The structure of Cry3Bb is similar to that of Cry3A (Li et al.,
1991). It consists of 5825 protein atoms from 588 residues (amino
acids 64-652) forming three discrete domains (FIG. 1). A total of
251 water molecules have been identified in the Cry3Bb structure
(FIG. 2). Domain 1 (residues 64-294) is a seven helical bundle
formed by six helices twisted around the central helix, .alpha.5
(FIG. 3). The amino acids forming each helix are listed in FIG. 4.
Domain 2 (residues 295-502) contains three antiparallel
.beta.-sheets (FIG. 5A and FIG. 5B). Sheets 1 and 2, each composed
of 4 .beta. strands, from the distinctive "Greek key" motif. The
outer surface of sheet 3, composed of 3 .beta. strands, makes
contact with helix .alpha.7 of domain 1. FIG. 6 lists the amino
acids comprising each .beta. strand in domain 2. A small .alpha.
helix, .alpha.8 which follows .beta. strand 1, is also included in
domain 2. Domain 3 (residues 503-652) has a "jelly roll"
.beta.-barrel topology which has a hydrophobic core and is nearly
parallel to the .alpha. and perpendicular to the c axes of the
lattice (FIG. 7A and FIG. 7B). The amino acids comprising each
.beta. strand of domains 3 are listed in FIG. 8.
The monomers of Cry3Bb in the crystal form a dimeric quaternary
structure along a two-fold axis parallel to the .alpha. axis (FIG.
9A and FIG. 9B). helix .alpha.6 lies in a cleft formed by the
interface of domain 1 and domains 1 and 3 of its symmetry related
molecule. There are numerous close hydrogen bonding contacts along
this surface, confirming the structural stability of the dimer.
5.2 Example 2
Precipitation of Cry3Bb.60
B. thuringiensis EG7231 was grown through sporulation in C2 medium
with chloramphenicol (Cml) selection. The solids form this culture
were recovered by centrifugation and washed with water. The toxin
was purified by recrystallization from 4.0M NaBr (Cody et al.,
1992). The purified Cry3Bb was solubilized in 10 ml of 50 mM
KOH/100 mg Cry3Bb and buffered to pH 9.0 with 100 mM CAPS (pH 9.0).
The soluble toxin was treated with trypsin at a weight ratio of 50
mg toxin to 1 mg trypsin. After 20 min of trypsin digestion the
predominant protein visualized by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) was 60 kDa. Further digestion of the
60-kDa toxin was not observed. FIG. 4 illustrates the
Coomassie-stained Cry3Bb and Cry3Bb.60 following SDS-PAGE.
5.3 Example 3
Purification and Sequence of Cry3Bb.60
Cry3Bb.60 was electrophoretically purified by SDS-PAGE and
electroblotted to Immobilon-.RTM. (Millipore) membrane by semi-dry
transfer at 15V for 30 min. The membrane was then washed twice with
water and stained with 0.025% R-250, 40% methanol. To reduce the
background, the blot was destained with 50% methanol until the
stained protein bands were visible. The blot was then air dried,
and the stained Cry3Bb.60 band was cut out of the membrane. This
band was sent to the Tufts University Sequencing Laboratory
(Boston, MA) for N-terminal sequencing. The
experimentally-determined N-terminal amino acid sequence is shown
in Table 7 beside the known amino acid sequence starting at amino
acid residue 160.
TABLE-US-00007 TABLE 7 AMINO ACID SEQUENCE OF THE N-TERMINUS OF
CRY3BB.60 AND COMPARISON TO THE KNOWN SEQUENCE OF CRY3BB Deduced
Sequence Known Sequence Residue # S S 160 K K 161 R R 262 S S 163 Q
Q 164 D D 165 R R 166
5.4 Example 4
Bioactivity of Cry3Bb.60
Cry3Bb was prepared for bioassay by solubilization in a minimal
amount of 50 mM KOH, 10 ml per 100 mg toxin, and buffered to pH 9.0
with 100 mM CAPS, pH 9.0. Cry3Bb.60 was prepared as described in
Example 1. Both preparations were kept at room temperature 12 to 16
hours prior to bioassay. After seven days the mortality of the
population was determined and analyzed to determine the lethal
concentration of each toxin. These results are numerized in Table
8.
TABLE-US-00008 TABLE 8 BIOACTIVITY OF CRY3BB AND CRY3BB.60 AGAINST
THE SOUTHERN CORN ROOTWORM (DIABIOTICA UNDECIMPUNCTATA) LC.sub.50
mg/well 95% C.I. Cry3Bb 24.09 15-39 Cry3Bb.60 6.72 5.25-8.4
5.5 Example 5
Ion-channel Formation by Cry3Bb AND CryB2.60
Cry3Bb.60 and Cry3Bb were evaluated for their ability to form ion
channels in planar lipid bilayers. Bilayers of phosphatidylcholine
were formed on Teflon.RTM. supports over a 0.7-mm hole. A bathing
solution of 3.5 ml 100 mM KOH, 10 mM CaCl.sub.2, 100 mM CAPS (pH
9.5) was placed on either side of the Teflon.RTM. partition. The
toxin was added to one side of the partition and a voltage of 60 mV
was imposed across the phosphatidylcholine bilayer. Any leakage of
ions through the membrane was amplified and recorded. An analysis
of the frequency of the conductances created by either Cry3Bb or
Cry3Bb.60 are illustrated in FIG. 5A and FIG. 5B. Cry3Bb.60 readily
formed ion channels whereas Cry3Bb rarely formed channels.
5.6 Example 6
Formation of High Molecular-weight Oligomers
Individual molecules of Cry3Bb or Cry3Bb.60 form a complex with
another like molecule. The ability of Cry3Bb to form an oligomer is
not reproducibly apparent. The complex cannot be repeatedly
observed to form under nondenaturing conditions. Cry3Bb.60 formed a
significantly greater amount of a higher molecular-weight complex
(.gtoreq.120 kDa) with other Cry3Bb.60 molecules. Oligomers of
Cry3Bb are demonstrated by the intensity of the Coomassie-stained
SDS polyacrylamide gel. Oligomerization is visualized on SDS-PAGE
by not heating samples prior to loading on the gel to retain some
nondenaturated toxin. These data suggest that Cry3Bb.60 more
readily forms the higher order complex than Cry3Bb alone.
Oligomerization is also observed by studying the conductance
produced by these molecules and the time-dependent increase in
conductance. This change in conductance can be attributed to
oligomerization of the toxin.
5.7 Example 7
Design Method 1: Identification and Alteration of
Protease-sensitive Sites and Proteolytic Processing
It has been reported in the literature that treatment of Cry3A
toxin protein with trypsin, an enzyme that cleaves proteins on the
carboxyl side of available lysine and arginine residues, yields a
stable cleavage product of 55 kDa from the 67 kDa native protein
(Carroll et al., 1989). N-terminal sequencing of the 55 kDa product
showed cleavage occurs at amino acid residue R158. The truncated
Cry3A protein was found to retain the same level of insecticidal
activity as the native protein. Cry3Bb toxin protein was also
treated with trypsin. After digestion, the protein size decreased
from 68 kDa, the molecular weight of the native Cry3Bb toxin, to 60
kDa. No further digestion was observed. N-terminal, sequencing
revealed the trypsin cleavage site of the truncated toxin
(Cry3Bb.60) to be amino acid R159 in l.alpha.3,4 of Cry3Bb.
Unexpectedly, the bioactivity of the truncated Cry3Bb toxin was
found to increase.
Using this method, protease digestion of a B. thuringiensis toxin
protein, a proteolytically sensitive site was identified on Cry3Bb,
and a more highly active form of the protein (Cry3Bb.60) was
identified. Modifications to this proteolytically-sensitive site by
introducing an additional protease recognition site also resulted
in the isolation of a biologically more active protein. It is also
possible that removal of other protease-sensitive site(s) may
improve activity. Proteolytically sensitive regions, once
identified, may be modified or utilized to produce biologically
more active toxins.
5.7.1 Cry3Bb.60
Treatment of solubilized Cry3Bb toxin protein with trypsin results
in the isolation of a stable, truncated Cry3Bb toxin protein with a
molecular weight of 60 kDa (Cry3Bb.60). N-terminal sequencing of
Cry3Bb.60 shows the trypsin-sensitive site to be R159 in
l.alpha.3,4 of the native toxin. Trypsin digestion results in the
removal of helices 1-3 from their native Cry3Bb but also increases
the activity of the toxin against SCRW larvae approximately
four-fold.
Cry3Bb.60 is a unique toxin with enhanced insecticidal use over the
parent Cry3Bb. Improved biological activity, is only one parameter
that distinguishes it as a new toxin. Aside from the reduced size,
Cry3Bb.60 is also a more soluble protein. Cry3Bb precipitates from
solution at pH 6.5 while Cry3Bb.60 remains in solution from pH 4.5
to pH 12. Cry3Bb.60 also forms ion channels with greater frequency
than Cry3Bb.
Cry3Bb.60 is produced by either the proteolytic removal of the
first 159 amino acid residues, or the in vivo production of this
toxin, by bacteria or plants expressing the gene for Cry3Bb.60,
that is, the Cry3Bb gene without the first 483 nucleotides.
In conclusion, Cry3Bb.60 is distinct from Cry3Bb in several
important ways: enhanced insecticidal activity; enhanced range of
solubility; enhanced ability to form channels; and reduced
size.
5.7.2 EG11221
Semi-random mutagenesis of the trypsin-sensitive l.alpha.3,4 region
of Cry3Bb resulted in the isolation of Cry3Bb.11221, a designed
Cry3Bb protein that exhibits over a 6-fold increase in activity
against SCRW larvae compared to WT. Cry3Bb.11221 has 4 amino acid
changes in the l.alpha.3,4 region. One of these changes, L158R,
introduces an additional trypsin site adjacent to R159, the
proteolytically sensitive site used to produce Cry3Bb.60 (example
4.1.1). Cry3Bb.11221 is produced by B. thuringiensis as a full
length toxin protein but is presumably digested by insect gut
protease to the same size as Cry3Bb.60 (see Cry3A results from
Carroll et al., 1989). The additional protease recognition site may
make the l.alpha.3,4 region even more sensitive to digestion,
thereby increasing activity.
5.8 Example 8
Design Method 2: Determination and Manipulation of Bound Water
There are several ways that water molecules can associate with a
protein, including surface water that is easily removed and bound
water that is more difficult to extract (Dunitz, 1994; Zhang and
Matthews, 1994). The function of bound water has been the subject
of significant academic extrapolation, but the precise function has
little experimental validation. Some of the most interesting bound
or structural water is the water that precipitates in the protein
structure from inside the protein itself.
The occupation of a site by a water molecule can indicate a stable
pocket within a protein or a looseness of packing created by
water-mediated salt bridges and hydrogen bonding to water. This can
reduce the degree of bonding between amino acids, possibly making
the region more flexible. A different amino acid sequence around
that same site could result in better packaging, collapsing the
pocket around polar or charged amino acids. This may result in
decreased flexibility. Therefore, the degree of hydration of a
region of a protein may determine the flexibility or mobility of
that region, and manipulation of the hydration may alter the
flexibility. Methods of increasing the hydration of a water-exposed
region include increasing the number of hydrophobic residues along
that surface. It is taught in the art that exposed hydrophobic
residues require significantly more water to hydrate than
hydrophilic residues (CRC Handbook of Chemistry and Physics, CRC
Press, Inc.). It is not taught, however, that by doing this,
improvements to the biological activity of a protein can be
achieved.
Structural water has not previously been identified in B.
thuringiensis .delta.-endotoxins including Cry3Bb. Furthermore,
there are no reports of the function to this structural water in
.delta.-endotoxins or bacterial toxins. In the analysis of Cry3Bb,
it was observed that a collection of water molecules are located
around l.alpha.3,4, a site defined by the inventors as important
for improvement of bioactivity. The loop l.alpha.3,4 region is
surface exposed and may define a hinge in the protein permitting
either removal or movement of the first three helices of domain 1.
The hydration found around this region may impart flexibility and
mobility to this loop. The observation of structural water all the
l.alpha.3,4 site provided an analytical tool for further structure
analysis. If this important site is surrounded by water, then other
important sites may also be completely or partially surrounded by
water. Using this insight, structural water surrounding helices 5
and 6 was then identified. This structural water forms a column
through the protein, effectively separating helices 5 and 6 from
the rest of the molecule. The structure of Cry3A and Cry3Bb suggest
that helices 5 and 6 are tightly associated, bound together by Van
de Waals interactions. Alone, helix 5 from Cry3A, although
insufficient for biological activity, has been demonstrated to have
the ability to form ion channels in an artificial membrane (Gazit
and Shai, 1993). The ion channels formed by elix 5 are 10-fold
smaller than the channels of the full length toxin suggesting that
significantly more toxin structure is required for the full-sized
ion channels. In Cry3Bb, helix 5 as part of a cluster of a helixes
(domain 1) has been found to form ion channels (Von Tersch et al.,
1994). Unpublished experimental observations by the inventors
demonstrate that helix 6 also crossed the biological membrane.
Helices 5 and 6, therefore, are the putative channel-forming
helices necessary for toxicity.
The hydration around these helices may indicate that flexibility of
this region is necessary for toxicity. It is conceivable,
therefore, that if it were possible to improve the hydration around
helices 5 and 6, one could create a better toxin protein. Care must
be taken, however, to avoid creating continuous hydrophobic
surfaces between helices 5-6 and any other part of the protein
which could, by hydrophobic interactions, act to restrict movement
of the mobile helices. The mobility of helices 5 and 6 may also
depend on the flexibility of the loops attached to them as well as
on other regions of the Cry3Bb molecule, particularly in domain 1,
which may undergo conformational changes to allow insertion of the
2 helices into the membrane. Altering the hydration of these
regions of the protein may also affect its bioactivity.
5.8.1 Cry3Bb.11032
A collection of bound water residues indicated the relative
flexibility of the l.alpha.3,4 region. The flexibility of this loop
can be increased by increasing the hydration of the region by
substituting relatively hydrophobic residues for the exposed
hydrophilic residues. An example of an improved, designed protein
having this type of substitution is Cry3Bb.11032. Cry3Bb.11032 has
the amino acid change D165G; glycine is more hydrophobic than
aspartate (Kyte and Doolittle hydrophobicity score of -4.0 vs. -3.5
for aspartate). Cry3Bb.11032 is approximately 3 times more active
than WT Cry3Bb.
5.8.2 Cry3Bb.11051
To increase the hydration of the l.alpha.4,5 region of Cry3Bb,
glycine was substituted for the surface exposed residue K189.
Glycine is more hydrophobic than lysine (Kyte and Doolittle
hydrophobicity score of -0.4 vs. -3.9 for lysine) and may result in
an increase in bound water. The increase in bound water may impart
greater flexibility to the loop region which precedes the
channel-forming helix, .alpha.5. The designed Cry3Bb protein with
the K189G change, Cry3Bb.11051, exhibits a 3-fold increase in
activity compared to WT Cry3Bb.
5.8.3 Alterations to L.alpha.7,.beta.1 (Cry3Bb.11241 and 11242)
Amino acid changes made in the surface-exposed loop connecting
.alpha.-helix 7 and .beta.-strand (l.alpha.7,.beta.1) resulted in
the identification of 2 altered Cry3 Bb proteins with increased
bioactivities, Cry3Bb11241 and Cry3Bb.11242. Analysis of the
hydropathy index of 2 of these proteins over the 20 amino acid
sequence 281-300, inclusive of the l.alpha.7,.beta.1 region, reveal
that the amino acid substitutions in these proteins have made the
l.alpha.7,.beta.1 region much more hydrophobic. The grand average
of hydropathy value (GRAVY) was determined for each protein
sequence using the PC\GENE.RTM. (IntelliGenetics, Inc., Mountain
View, Calif., release 6.85) protein sequence analysis computer
program, SOAP, and a 7 amino acid interval. The SOAP program is
based on the method of Kyte and Doolittle (1982). The increase in
hydrophobicity of the l.alpha.7,.beta.1 region for each protein may
increase the hydration of the loop and therefore, the flexibility.
The altered proteins, their respective amino acid changes,
fold-increases over WT bioactivity, and GRAVY values are listed in
Table 9.
TABLE-US-00009 TABLE 9 HYDROPATHY VALUES FOR THE Ln7, .beta.1
REGION OF CRY3BB AND 2 DESIGNED CRY3BB PROTEINS SHOWING INCREASED
SCRW BIOACTIVITY Fold Increase in GRAVY Cry3Bb* Amino Acid
Bioactivity Over (Amino Acids Protein Changes WT 281-300) wildtype
-- -- 4.50 Cry3Bb.11241 Y287F, D288N, 2.6x 10.70 R290L Cry3Bb.11242
R290V 2.5x 8.85
5.8.4 Alteations of L.beta.1,.alpha.8 (Cry3Bb,11228, Cry3Bb.11229,
Cry3Bb.11230, Cry3Bb.11233, Cry3Bb.11236, Cry3Bb.11237,
Cry3Bb.11238, and Cry3Bb.11239)
The surface-exposed loop between .beta.-strand 1 and .alpha.-helix
8(1.beta.,.alpha.8) defines the boundary between domains 1 and 2 of
Cry3Bb. The introduction of semi-random amino acid changes to this
region resulted in the identification of several altered Cry3Bb
proteins with increased bioactivity. Hydropathy index analysis of
the amino acid substitutions found in the altered proteins shows
that the changes have made the exposed region more hydrophobic
which may result in increased hydration and flexibility. Table 10
lists the altered proteins, their respective amino acid changes and
fold increases over WT Cry3Bb and the grand average of hydropathy
value (GRAVY) determined using the PC\GENE.RTM. (IntelliGenetics,
Inc., Mountain View, Calif., release 6.85) protein sequence
analysis program, SOAP, over the 20 amino acid sequence 305-324
inclusive of 1.beta.1,.alpha.8, using a 7 amino acid interval.
TABLE-US-00010 TABLE 10 HYDROPATHY VALUES FOR THE L.beta.1, .mu.8
REGION OF CRY3BB AND 8 DESIGNED CRY3BB* PROTEINS SHOWING INCREASED
SCRW BIOACTIVITY Fold Increase in GRAVY Cry3Bb* Amino Acid
Bioactivity Over (Amino Acids Protein Changes Wild Type 305-324)
wildtype -- -- 0.85 Cry3Bb.11228 S311I, N313T, 4.1x 4.35 E317K
Cry3Bb.11229 S311T, E317K, 2.5x 2.60 Y318C Cry3Bb.11230 S311A,
L312V, 4.7x 3.65 Q316W Cry3Bb.11233 S311A, Q316D 2.2x 2.15
Cry3Bb.11236 S311I 3.1x 3.50 Cry3Bb.11237 S311I, N313H 5.4x 3.65
Cry3Bb.11238 N313V, T314N, 2.6x 9.85 Q316M, B317V Cry3Bb.11239
N313R, L315P, 2.8x 3.95 Q316L, E317A
5.8.5 Cry3Bb.11227, Cry3Bb.11241 and Cry3Bb.11242
Amino acid Q238, located in helix 6 of Cry3Bb, has been identified
as a residue that, by its large size and hydrogen bonding to R290,
blocks complete hydration of the space between helix 6 and helix 4.
Substitution of R290 with amino acids that do not form hydrogen
bonds or that have side chains that can not span the physical
distance to hydrogen bond with Q238 may result in increased
hydration around Q238. Q238, unable to hydrogen bond to R290, may
now bind water. This may increase the flexibility of the
channel-forming region. Designed proteins Cry3Bb.11227 (R290N),
Cry3Bb.11241 (R290L) and Cry3Bb.11242 (R290V) show increased
activities of approximately 2-fold, 2.6-fold and 2.5-fold,
respectively, against SCRW larvae compared to WT.
5.9 Example 9
Design Method 3: Manipulation of Hydrogen Bonds Around Mobile
Regions
Mobility of regions of a protein may be required for activity. The
mobility of the .alpha.5,6 region, the putative channel-forming
region of Cry3Bb, may be improved by decreasing the number of
hydrogen bonds, including salt bridges (hydrogen bonds between
oppositely charged amino acid side chains), between helices 5-6 and
any other part of the molecule or dimer structure. These hydrogen
bonds may impede the movement of the two helices. Decreasing the
number of hydrogen bonds and salt bridges may improve biological
activity. Replacement of hydrogen-bonding amino acids with
hydrophobic residues must be done with caution to avoid creating
continuous hydrophobic surfaces between helices 5-6 and any other
part of the dimer. This may decrease mobility by increasing
hydrophobic surface interactions.
5.9.1 Cry3Bb.11222 and Cry3Bb.11223
Tyr230 is located on helix 6 and, in the quaternary dimer structure
of Cry3Bb, this amino acid is coordinated with Tyr230 from the
adjacent molecule. Three hydrogen bonds are formed between the two
helices 6 in the two monomers because of this single amino acid. In
order to improve the flexibility of helices 5-6, the helices
theoretically capable of penetrating the membrane and forming an
ion channel, the hydrogen bonds across the dimer were removed by
changing this amino acid and a corresponding increase in biological
activity was observed. The designed Cry3Bb proteins, Cry3Bb.11222
and Cry3Bb.EG11223, show a 4-fold and 2.8-fold increase in SCRW
activity, respectively, compared to WT.
5.9.2 Cry3Bb.11051
Designed Cry3Bb protein Cry3Bb.11051 has amino acid change K189G in
l.alpha.4,5 of domain 1. In the WT Cry3Bb structure, the exposed
side chain of K189 is close enough to the exposed side change of
E123, located in l.dbd.2b,3, to form hydrogen bonds. Substitution
of K189 with glycine, as found in this position in Cry3A, removes
the possibility of hydrogen bond formation at this site and results
in a protein with a bioactivity three-fold greater than WT
Cry3Bb.
5.9.3 Cry3Bb.11227, Cry3Bb.11241 and Cry3Bb.11242
Amino acid Q238, located in helix 6 of Cry3Bb, has been identified
as a residue that, by its large size and hydrogen bonding to R290,
blocks complete hydration of the space between helix 6 and helix 4.
Substitution of R290 with amino acids that do not form hydrogen
bonds or that have side chains that can not span the physical
distance to hydrogen bond with Q238 may increase the flexibility of
the channel-forming region. Designed proteins Cry3Bb.11227 (R290N),
Cry3Bb.11241 (R290L), and Cry3Bb.11242 (R290V) show increased
activities of approximately 2-fold, 2.6-fold and 2.5-fold,
respectively, against SCRW larvae compared to WT
5.10 Example 10
Design Method 4: Loop Analysis and Loop Design Around Flexible
Helices
Loop regions of a protein structure may be involved in numerous
functions of the protein including, but not limited to, channel
formation, quaternary structure formation and maintenance, and
receptor binding. Cry3Bb is a channel-forming protein. The
availability of the ion channel-forming helices of
.delta.-endotoxins to move into the bilayer depend upon the absence
of forces that hinder the process. One of the forces possibly
limiting this process is the state hindrance of amino acid side
chains in loop regions around the critical helices. The literature
suggests that in at least one other bacterial toxin, not a B.
thuringiensis toxin, the toxin molecule opens up or, in scientific
terms, loses some of the quaternary structure to expose a
membrane-active region (Cramer et al., 1990). This literature does
not teach how to improve the probability of this event occurring
and it is not known if B. thuringienis toxins use this same process
to penetrate the membrane. Reducing the steric hindrance of the
amino acid side chains in these critical regions by reducing size
or altering side chain positioning with the corresponding increase
in biological activity was the invention step.
5.10.1 Analysis of the Loop Between Helices 3 and 4
(Cry3Bb.11032)
The inventors have discovered that the first three helices of
domain one could be cleaved from the rest of the toxicity by
proteolytic digestion of the loop between helices .alpha.3 and
.alpha.4 (Cry3Bb.60). Initial efforts to truncate the cry3Bb gene
to produce this shortened, though more active Cry3Bb molecule,
failed. For unknown reasons, B. thuringiensis failed to synthesize
this 60-kDa molecule. It was then reasoned that perhaps the first
three helices of domain 1 did not have to be proteolytically
removed, or equivalently, the protein did not have to be
synthesized in this truncated form to take advantage of the
Cry3Bb.60 design. It was observed that the protein Cry3A had a
small amino acid near the l.alpha.3,4 that might impart greater
flexibility in the loop region thereby permitting the first three
helices of domain 1 to move out of the way, exposing the
membrane-active region. By designing a Cry3Bb molecule with a
glycine residue near this loop, the steric hindrance of residues in
the loop might be lessened. The redesigned protein, Cry3Bb.11032,
has the amino acid change D165G, which replaces the larger
aspartate residue (average mass of 115.09) with the smallest amino
acid, glycine (average mass of 57.05). The activity of Cry3Bb.11032
is approximately 3-fold greater than that of the WT protein. In
this way, the loop between helices .alpha.3 and .alpha.4 was
rationally redesigned with a corresponding increase in the
biological activity.
5.10.2 Cry3Bb.11051
The loop region concentrating helices .alpha.4 and .alpha.5 in
Cry3Bb must be flexible so that the channel-forming helices
.alpha.5-.alpha.6 can penetrate into the membrane. It was noticed
that Cry3A has a glycine residue in the middle of this loop that
may impart greater flexibility. The corresponding change, K189G,
was made in Cry3Bb and the resulting, designed protein,
Cry3Bb.11051, exhibits a 3-fold increase in activity against SCRW
larvae compare to WT Cry3Bb.
5.10.3 Analysis of the Loop Between .beta.-strand 1 and Helix 8
(Cry3Bb.11228, Cryb 3Bb.11229, Cry3Bb.11230, Cry3Bb.11232,
Cry3Bb.11233, Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238, and
Cry3Bb.11239)
The loop region located between .beta. strand 1 of domain 2 and
.alpha. helix 8 in domain 2 is very close to the loop between
.alpha. helices 6 and 7 in domain 1. Some of the amino acids side
chains of 1.beta.1,.alpha.8 appear as though they may sterically
impede movement of l.alpha.6,7. Since the l.alpha.7, must be
flexible for the channel-forming helices .alpha.5-.alpha.6 to
insert into the membrane, it was thought that re-engineering this
loop may change the positioning of the side chains resulting in
less steric hindrance. This was accomplished creating proteins with
increased biological activities ranging from 2.2 to 5.4 times
greater than WT. These designed toxin proteins and their amino acid
changes are listed in Table 2 as Cry3Bb.11228, Cry3Bb.11229,
Cry3Bb.11230, Cry3Bb.11232, Cry3Bb.11233, Cry3Bb.11236,
Cry3Bb.11237, Cry3Bb.11238, and Cry3Bb.11239.
5.10.4 Analysis of the Loop Between Helix 7 and .beta.-strand 1
(Cry3Bb.11227, Cry3Bb.11234, Cry3Bb.11241, Cry3Bb.11242, and
Cry3Bb.11036)
If Cry3Bb is similar to a bacterial toxin which must open up to
expose a membrane active region for toxicity, it is possible that
other helices in addition to the channel-forming helices must also
change positions. It was reasoned that, if helices
.alpha.5-.alpha.6 insert into the membrane, than helix .alpha.7 may
have to change positions also. It was shown in example 4.4.3 that
increasing flexibility between helix .alpha.6 and .alpha.7 can
increase activity, greater flexibility in the loop following helix
.alpha.7,1.alpha.7,.beta.1 may also increase bioactivity.
Alterations to the l.alpha.7,.beta.1 region of Cry3Bb resulted in
the isolation of several proteins with increased activities ranging
from 1.9 to 4.3 times greater than WT. These designed proteins are
listed in Table 7 as CryBb.11227, Cry3Bb.11234, Cry3Bb.11241,
Cry3Bb.11242, and Cry3Bb.11036.
5.11 Example 11
Design Methods 5: Loop Design Around .beta. Strands and .beta.
Sheets
Loop regions of a protein structure may be involved in numerous
functions of the protein including, but not limited to, channel
formation, quaternary structure formation and maintenance, and
receptor binding. A binding surface is often defined by a number of
loops, as is the case with immunoglobulin G (IgG) (see Branden and
Tooze, 1991, for review). What can not be determined at this point,
however, is what loops will be important for receptor interactions
just by looking at the structure of the protein in question. Since
a receptor has not been identified for Cry3Bb, it is not even
possible to compare the structure of Cry3Bb with other proteins
that have the same receptor for structural similarities. To
identify Cry3Bb loops that contribute to receptor interactions,
random mutagenesis was performed on surface-exposed loops.
As each loop was altered, the profile of the overall bioactivities
of the resultant proteins were examined and compared. The loops,
especially in domain 2 which appears to be unnecessary for channel
activity, fall into two categories: (1) loops that could be altered
without much change in the level of bioactivity of the resultant
proteins and (2) loops where alterations resulted in overall loss
of resultant protein bioactivity. Using this design method, it is
possible to identify several loops important for activity.
5.11.1 Analysis of Loop .beta.2,3
Semi-random mutagenesis of the loop region between .beta. strands 2
and 3 resulted in the production of structurally stable toxin
proteins with significantly reduced activities against SCRW larvae.
The 1.beta.2,3 region is highly sensitive to amino acid changes
indicating that specific amino acids or amino acid sequences are
necessary for toxin protein activity. It is conceivable, therefore,
that specific changes in the 1.beta.2,3 region will increase the
binding and, therefore, the activity of the redesigned toxin
protein.
5.11.2 Analysis of Loop .beta.6,7
Semi-random mutations, introduced to the loop region between .beta.
strands 6 and 7 resulted in structurally stable proteins with an
overall loss of SCRW bioactivity. The 1.beta.6,7 region is highly
sensitive to amino acid changes indicating that specific amino
acids or amino acid sequences are necessary for toxin protein
activity. It is conceivable, therefore, that specific changes in
the 1.beta.6,7 region will increase the binding and, therefore, the
activity of the redesigned toxin protein.
5.11.3 Analysis of Loop .beta.10,11
Random mutations to the loop region between .beta. strands 10 and
11 resulted in proteins having an overall loss of SCRW bioactivity.
Loop .beta.10,11 is structurally close to and interacts with loops
.beta.2,3 and .beta.6,7. Specific changes to individual residues
within the 1.beta.10,11 region may also result in increased
interaction with the insect membrane, increasing the bioactivity of
the toxin protein.
5.11.4 Cry3Bb.11095
Loops .beta.2,3, .beta.6,7 and .beta.10,11 have been identified as
important for bioactivity for Cry3Bb. The 3 loops are
surface-exposed and structurally close together. Amino acid Q348 in
the WT structure, located in .beta.-strand 2 just prior to
1.beta.2,3 doe not form any intramolecular contacts. However,
replacing Q348 with arginine (Q348R) results in the formation of 2
new hydrogen-bonds between R348 and the backbone carbonyls of R487
and R488, both located in 1.beta.10,11. The new hydrogen bonds may
act to stabilize the structure formed by the 3 loops. The designed
protein carrying this change, Cry3Bb.11095, is 4.6-fold more active
than WT Cry3Bb.
5.12 Example 12
Design Method 6: Identification and Re-design of Complex
Electrostatic Surfaces
Interactions of proteins include hydrophobic interactions (e.g.,
Van der Waals forces), hydrophilic interactions, including those
between opposing charges on amino acid side chains (salt bridges),
and hydrogen bonding. Very little is known about .delta.-endotoxin
and receptor interactions. Currently, there are not literature
reports identifying the types of interactions that predominate
between B. thuringiensis toxins and receptors.
Experimentally, however, it is important to increase the strength
of the B. thuringiensis toxin-receptor interaction and not permit
the precise determination of the chemical interaction to stand in
the way of improving it. To accomplish this, the electrostatic
surface of Cry3Bb was defined by solving the Poisson-Boltzman
distribution around the molecule. Once this electrically defined
surface was solved, it could then be inspected for regions of
greatest diversity. It was reasoned that these electrostatically
diverse regions would have the greatest probability of
participating in the specific interactions between the B.
thuringiensis toxin proteins and the receptor, rather than more
general and non-specific interactions. Therefore, these regions
were chosen for redesign, continuing to increase the electrostatic
diversity of the regions. In addition, examination of the
electrostatic interaction around the putative channel forming
region of the toxin created insights for redesign. This includes
identification of an electropositive residue in an otherwise
negatively charged conduit (see example 4.6.1).
5.12.1 R290 (Cry3Bb.11227, Cry3Bb.11241, and Cry3Bb.11242)
Examination of the Cry3Bb dimer interfaces along the domain 1 axis
suggested that a pore or conduit for cations might be formed
between the monomers. Electrostatic examination of this axis lent
additional credibility to this suggestion. In fact, the
hypothetical conduit is primarily negatively charged, an
observation consistent with the biophysical analysis of
cation-selective, .delta.-endotoxin channels. If a cation channel
were formed along the axis of the dimer, then the cation could move
between the monomers relatively easily with only one significant
hurdle. A positively charged arginine residue (R290) lies in the
otherwise negatively charged conduit. This residue could impede the
cation movement through be channel. Based on this analysis, R290
was changed to unchanged residues. The bioactivity of redesigned
proteins Cry3Bb.11227 (R290N), Cry3Bb.11241 (R290L) and
Cry3Bb.11242 (R290V) was improved approximately 2-fold, 2.6-fold
and 2.5-fold, respectively.
5.12.2 Cry3Bb.60
Trypsin digestion of solubilized Cry3Bb yields a stable, truncated
protein with a molecular weight of 60 kDa (Cry3Bb.60). Trypsin
digestion occurs on the carboxyl side of residue R159, effectively
removing helices 1 through 3 from the native Cry3Bb structure. The
cleavage of the first 3 helices exposes an electrostatic surface
different than those found in the native structure. The new surface
has a combination of hydrophobic, polar and charged characteristics
that may play a role in membrane interactions. The bioactivity of
Cry3Bb.60 is 3.6-fold greater than that of WT Cry3Bb.
5.13 Example 13
Design Method 7: Identification and Removal of Metal Binding
Sites
The literature teaches that the in vitro behavior of B.
thuringiensis toxins can be increased by chelating divalent cations
from the experimental system (Crawford and Harvey 1988). It was not
known, however, how these divalent cations inhibited the in vitro
activity. Crawford and Harvey (1988) demonstrated that the short
circuit current across the midgut was more severely inhibited by B.
thuringiensis in the presence of EDTA, a chelator of divalent ions,
than in the absence of this agent, thus suggesting that this step
in the mode of action of B. thuringiensis could be potentiated by
removing divalent ions. Similar observations were made using
black-lipid membranes and measuring an increase in the current
created by the .delta.-endotoxins in the presence of EDTA to
chelate divalent ions. There were at least three possible
explanations for these observations. The first explanation could be
that the divalent ions are too large to move through a ion channel
more suitable for monovalent ions, thereby blocking the channel.
Second, the divalent ions may cover the protein in the very general
way, thereby buffering the charge interactions required for toxin
membrane interaction and limiting ion channel activity. The third
possibility is that a specific metal binding site exists on the
protein and, when occupied by divalent ions, the performance of the
ion channel is impaired. Although the literature could not
differentiate the value of one possibility over another, the third
possibility led to an analysis of the Cry3Bb structure searching
for a specific metal binding site that might alter the probability
that a toxin could form an ion channel.
5.13.1 H231 (Cry3Bb.11222, Cry3Bb.11224, Cry3Bb.11225, and
Cry3Bb.11226)
A putative metal binding site is formed in the Cry3Bb dimer
structure by the H231 residues of each monomer. The H231 residues,
located in helix a6, lie adjacent to each other and close to the
axis of symmetry of the dimer. Removal of this site by replacement
of histidine with other amino acids was evaluated by the absence of
EDTA-dependent ion channel activity. The bioactivities of the
designed toxin proteins, Cry3Bb.11222, Cry3Bb.11224, Cry3Bb.11225
and Cry3Bb.11226, are increased 4-, 5-, 3.6- and 3-fold,
respectively, over that of WT Cry3Bb. Their respective amino acid
changes are listed in Table 2.
5.14 Example 14
Design Method 8: Alteration of Quaternary Structure
Cry3Bb can exist in solution as a dimer similar to a related
protein, Cry3A (Walters et al., 1992). However, the importance of
the dimer to biological activity is not known because the toxin as
a monomer or as a higher order structure has not been seriously
evaluated. It is assumed that specific amino acid residues
contribute to the formation and stability of the quaternary
structure. Once a contributing residue is identified, alterations
can be made to diminish or enhance the effect of that residue
thereby affecting the interaction between monomers. Channel
activity is a useful way, but by no means the only way, to assess
quaternary structure of Cry3Bb and its derivatives. It has been
observed that Cry3Bb creates gated conductances in membranes that
grow in size with time, ultimately resulting in large pores in the
membrane (the channel activity of WT Cry3Bb is described in Section
12.1). It also has been observed that Cry3A forms a more stable
dimer than Cry3Bb and coincidentally forms higher level
conductances faster (FIG. 10). This observation led the inventors
to propose that oligomerization and ion channel formation
(conductance size and speed of channel formation) were related.
Based on this observation Cry3Bb was re-engineered to make larger
and more stable oligomers at a faster rate. It is assumed in this
analysis that the rate of ion channel formation and growth mirrors
this process. It is also possible that changes in quaternary
structure may not affect channel activity alone or at all.
Alterations to quarternary structure may also affect receptor
interactions, protein processing in the insect gut environment, as
well as other aspects of bioactivity unknown.
5.14.1 Cry3Bb.11048
Comparative structural analysis of Cry3A and Cry3Bb led to the
identification of structural differences between the two toxins in
the ion channel-forming domain, specifically, an insertion of one
amino acid between helix 2a and helix 2b in Cry3Bb. Removal of this
additional amino acid in Cry 3B2, A104, and a D103E substitution,
as in Cry3A, resulted in loss of channel gating and the formation
of symmetrical pores. Once the pores are formed they remain open
and allow a steady conductance ranging from 25-139 pS. This
designed protein, Cry3Bb.11048, is 4.3 times more active than WT
Cry3Bb against CCRW larvae.
5.14.2 Oligomerization of Cry3Bb.60
Individual molecules of Cry3Bb or Cry3Bb.60 can form a complex with
another like molecule. Oligomerization of Cry3Bb is demonstrated by
SDS-PAGE, where sample are not heated in sample buffer prior to
loading on the gel. The lack of heat treatment allows some
nondenatured toxin to remain. Oligomerization is visualized
following Coomassie staining by the appearance of a band at 2 times
the molecular weight of the monomer. The intensity of the higher
molecular weight band reflects the degree of oligomerization. The
ability of Cry3Bb to form an oligomer is not reproducibly apparent.
The complex cannot be repeatedly observed to form. Cry3Bb.60,
however, forms a significantly greater amount of a higher molecular
weight complex (120 kDa). These data suggest that Cry3Bb.60 more
readily forms the higher order complex than Cry3Bb alone. Cry3Bb.60
also forms ion channels with greater frequency than WT Cry3Bb (see
Section 5.12.9).
5.14.3 Cry3Bb.11035
Changes were made in Cry3Bb to reflect the amino acid sequence in
Cry3A at the end of the l.alpha.3,4 and in the beginning of helix
4. These changes resulted in the designed protein, Cry3Bb.11035,
that, unlike wild type Cry3Bb, forms spontaneous channels with
large conductances. Cry3Bb.11035 is also approximately three times
more active against SCRW larvae than WT Cry3Bb. Cry3Bb.11035 and
its amino acid changes are listed in Table 10.
5.14.4 Cry3Bb.11032
Cry3Bb.11032 was altered at residue 165 in helix .alpha.4, changing
an asparate to glycine, as found in Cry3A. Cry3Bb.11032 is
three-fold more active than WT Cry3Bb. The channel activity of
Cry3Bb.11032 is much like Cry3Bb except when the designed protein
is artificially incorporated into the membrane. A 16-fold increase
in the initial channel conductances is observed compared to WT
Cry3Bb (see Section 5.12.2). This increase in initial conductance
presumably is due to enhanced quaternary structure, stability of
higher-order structure.
5.14.5 EG11224
In the WT Cry3Bb dimer structure, histidine, at position 231 in
domain 1, makes hydrogen bond contacts with D288 (domain 1), Y230
(domain 1), and, through a network of water molecules, also makes
contacts to D610 (domain 3), all of the opposite monomer. D610 and
K235 (domain 1) also make contact. Replacing the histidine with an
arginine, H231R, results, in one orientation, in the formation of a
salt bridge to D610 of the neighboring monomer. In a second
orientation, the contacts with D288 of the neighboring monomers, as
appear in the WT structure, are retained. In either orientation,
I231 does not hydrogen bond to Y230 of the opposite monomer but
does make contact with K235 which retains is contacts to K610 (V.
Cody, research communication). The shifting hydrogen bonds have
changed the interactions between the different domains of the
protein in the quaternary structure. Overall, fewer hydrogen bonds
exist between domains 1 of the neighboring monomers and a much
stronger bond has been formed between domains 1 and 3. Channel
activity was found to be altered. Cry3Bb.11224 produces small,
quickly gating channels like Cry3Bb. However, unlike WT Cry3Bb,
Cry3Bb.11224 does not exhibit, .beta.-mercaptoethanol-dependent
activation. Replacing H231 with arginine resulted in a designed
Cry3Bb protein, Cry3Bb.11224, exhibiting a 5-fold increse ion
bioactivity.
5.14.6 Cry3Bb.11226
Cry3Bb.11226 is similar to Cry3Bb.11224, discussed in Section
4.8.5, in that the histidine at position 231 has been replaced. The
amino acid change, H231T, results in the loss of
.beta.-mercaptoethanol dependent activation seen with WT Cry3Bb
(see Section 5.12.1). The replacement of H231, a putative metal
binding site, changes the interaction of regions in the quarternary
structure resulting in a different type of channel activity.
Cry3Bb.11226 is three-fold more active than WT Cry3Bb.
5.14.7 Cry3Bb.11221
Cry3Bb.11221 has been re-designed in the l.alpha.3,4 region of
Cry3Bb. The channels formed by Cry3Bb.11221 are much more well
resolved that the conductances formed by WT Cry3Bb (see Section
5.12.6). Cry3Bb.11221 exhibits a 6.4-fold increase in bioactivity
over that of WT Cry3Bb. The amino acid changes found in
Cry3Bb.11221 are listed in Table 2.
5.14.8 Cry3Bb.11242
The designed protein, Cry3Bb.11242, carrying the alteration R290V,
forms small conductances immediately which grow rapidly and
steadily to large conductances in about 3 min (see Section 5.12.7).
This is contrast to WT Cry3Bb channels which take 30-45 min to
appear and grow slowly over hours to large conductances.
Cry3Bb.11242 also exhibits a 2.5-fold increase in bioactivity
compared to WT Cry3Bb.
5.14.9 Cry3Bb.11230
Cry3Bb,11230, unlike WT Cry3Bb, forms well resolved channels with
long open states. These channels reach a maximum conductance of
3000 pS but do not continue to grow with time. Cry3Bb.11230 has
been re-designed in the 1.beta.,.alpha.8 region of Cry3Bb and
exhibits almost a 5-fold increase in activity against SCRW larvae
(Table 9) and a 5.4-fold increase against WCRW larvae (Table 10)
compared to WT Cry3Bb. The amino acid changes found in Cry3Bb.11230
are listed in Table 2.
5.15 Example 15
Design Method 9: Design of Structural Residues
The specific three-dimensional structure of a protein is held in
place by amino acids that may be buried or otherwise removed from
the surface of the protein. These structural determinants can be
identified by inspection of forces responsible for the surface
structure positioning. The impact of these structural residues can
then be enhanced to restrict molecular motion or diminished to
enhance molecular flexibility.
5.15.1 Cry3Bb.11095
Loops .beta.2,3, .beta.6,7 and .beta.10,11, located in domain 2 of
Cry3Bb, have been identified as important for bioactivity. The
three loops are surface-exposed and structurally close together.
Amino acid Q348 in the WT structure, located in .beta.-strand 2
just prior to 1.beta.2,3, does not form any intramolecular
contacts. However, replacing Q348 with arginine (Q348R) results in
the formation of 2 new hydrogen-bonds between R348 and the backbone
carbonyls of R487 and R488, both located in 1.beta.10,11. The new
hydrogen bonds may act to stabilize the structure formed by the
three loops. Certainly, the structure around R348 is more lightly
packed as determined by X-ray crystallography. The designed protein
carrying this change, Cry3Bb.11095, is 4.6-fold more active than WT
Cry3Bb.
5.16 Example 16
Design Method 10: Combinatorial Analysis and Mutagensis
Individual sites in the engineered Cry3Bb molecule can be used
together to create a Cry3Bb molecule with activity even greater
than the activity of any one site. This method has not been
precisely applied to any .delta.-endotoxin. It is also not obvious
that improvements in two sites can be pulled together to improve
the biological activity of the protein. In fact, data demonstrates
that improvements to 2 sites, when pulled together into a single
construct, do not necessarily further improve the biological
activity of Cry3Bb. In some cases, the combination resulted in
decreased protein stability and/or activity. Examples of proteins
with site combinations that resulted in improved activity compared
to WT Cry3Bb but decreased activity compared to 1 or more of the
"parental" proteins are Cry3Bb.11235, 11046, 11057 and 11058.
Cry3Bb.11082, which contains designed regions from 4 parental
proteins, retains the level of activity from the most active
parental strain (Cry3Bb.11230) but does not show an increase in
activity. These proteins are listed in Table 7. The following are
examples of instances where combined mutations having significantly
improved biological activity.
5.16.1 Cry3Bb.11231
Designed protein Cry3Bb.11231 contains the alterations found in
Cry3Bb.11224 (H231R) and Cry3Bb.11228 (changes in
1.beta.1,.alpha.8). The combination of amino acid changes found in
Cry3Bb.11231 results in an increase in bioactivity against SCRW
larvae of approximately 8-fold over that of WT Cry3B (Table 2).
This increase is greater than exhibited by either Cry3Bb.11224
(5.0.times.) or Cry3Bb.11228 (4.1.times.) alone. Cry3Bb.11231 was
also exhibits an 12.9-fold increase in activity compared to WT
Cry3Bb against WCRW larvae (Table 10).
5.16.2 Cry3Bb.11081
Designed Cry3Bb protein Cry3Bb.11081 was constructed by combining
the changes found in Cry3Bb.11032 and Cry3Bb.11229 (with the
exception of Y318C). Cry3Bb.11081 a 6.1-fold increase in activity
over WT Cry3Bb; a greater increase in activity than either of the
individual parental proteins, Cry3Bb.11032 (3.1-fold) and
Cry3Bb.11229 (2.5-fold).
5.16.3 Cry3Bb.11083
Designed Cry3Bb protein Cry3Bb.11083 was constructed by combining
the changes found in Cry3Bb.11036 and Cry3Bb.11095, Cry3Bb.11083
exhibits a 7.4-fold increase in activity against SCRW larvae
compared to W Cry3Bb; a greater increase than either Cry3Bb.11036
(4.3.times.) or Cry3Bb.11095 (4.6.times.). Cry3Bb.11083 also
exhibits a 5.4-fold increase in activity against WCRW larvae
compared to WT Cry3Bb (Table 10).
5.16.4 Cry3Bb.11084
Designed Cry3Bb protein Cry3Bb.11084 was constructed by combining
the changes found in Cry3Bb.11032 and the S311L change found in
Cry3Bb.11228. Cry3Bb.11084 exhibits a 7.2-fold increase in activity
over that of WT Cry3Bb; a greater than either Cry3Bb.11032
(3.1.times.) or Cry3Bb.11228 (4.1.times.).
5.16.5 Cry3Bb.11098
Designed Cry3Bb protein Cry3Bb.11098 was constructed to contain the
following amino acid changes: D165G, H231R, S311L, N313T, and
E317K. The nucleic acid sequence is given in SEQ ID NO:107, and the
encoded amino acid sequence is given in SEQ ID NO:108.
5.17 Example 17
Design Strategy 11: Alteration of Binding to Glycoproteins and to
WCRW Brush Border Membranes
While the identity of receptor(s) for Cry3Bb is unknown, it is
nonetheless important to increase the interaction of the toxin with
its receptor. One way to improve the toxin-receptor interaction
with knowing the identity of the receptor is to reduce or eliminate
non-productive binding to other biomolecules. The inventors have
observed that Cry3Bb binds non-specifically to bovine serum albumin
(BSA) that has been glycosylated with a variety of sugar groups,
but not to non-glycosylated BSA. Cry3A, which is not active on
Diabrotica species, shows similar but even greater binding to
glycosylated-BSA. Similarly, Cry3A shows greater binding to
immobilized WCRW brush border membrane (BBM) than does WT Cry3Bb,
suggesting that much of the observed binding is non-productive. It
was reasoned that the non-specific binding to WCRW BBM occurs via
glycosylated proteins, and that binding to both glycosylated-BSA
and WCRW BBM is non-productive in reaction pathway to toxicity.
Therefore reduction or elimination of that binding would lead to
enhanced binding to the productive receptor and to enhanced
toxicity. Potential binding sites for sugar groups were targeted
for redesign to reduce the non-specific binding of Cry3Bb to
glycoproteins and to immobilized WCRW BBM.
5.17.1 Cry3Bb.60
Cry3Bb-60, in which Cry3Bb has been cleaved at R159 in 1.alpha.3,4
shows decreased binding to glycosylated-BSA and decreased binding
to immobilized WCRW BBM. Cry3Bb60 shows a 3.6-fold increase in
bioactivity relative to WT Cry3Bb.
5.17.2 Alterations to 1.alpha.3,4 (Cry3Bb.11221)
Cry3Bb.11221 has been redesigned in the 1.alpha.3,4 region of
domain 1, which is the region in which Cry3Bb is cleaved to produce
Cry3Bb-60. Cry3Bb.11221 also shows decreased binding to both
glycosylated-BSA and immobilized WCRW BBM, and exhibits a 6.4-fold
increase in bioactivity over that of WT Cry3Bb. Together with data
for Cry3Bb.60 (section 5.17.1) these data suggest that this loop
region contributes substantially to non-productive binding of the
toxin.
5.17.3 Alteration to 1.beta.1,.alpha.8 (Cry3Bb.11228,11230, 11237
and 11231)
The 1.beta.1,.alpha.8 region of Cry3Bb has been re-engineered to
increase hydration (section 4.2.4) and enhance flexibility (section
4.4.3). Several proteins altered in this region,
Cry3Bb.11228,11230, and 11237 demonstrate substantially lower
levels of binding both glycosylated-BSA and immobilized WCRW BBM,
and also show between 4.1- and 4.5-fold increases in bioactivity
relative to WT Cry3Bb.
5.17.4 Binding Activity
The tendencies of Cry3Bb and some of its derivatives to bind to
glycosylated-BSA and to WCRW BBM were determined using a
BIAcore.TM. surface plasmon resonance biosensor. For
glycosylated-BSA binding, the glycosylated protein was immobilized
using standard NHS chemistry to a CM5 chip (BIAcore), and the
solubilized toxin was injected over the glycosylated-BSA surface.
To measure binding to WCRW BBM, brush border membrane vesicles
(BBMV) purified from WCRW midguts (English et al., 1991) were
immobilized on an HPA chip (BIAcore) then washed with either 10 mM
KOH or with 40 mM .beta.-octylglucoside. The solubilized toxin was
then injected over the resulting hybrid bilayer surface to detect
binding. Protein concentration were determined by Protein Dye
Reagent assay (BioRad) or BCA Protein Assay (Pierce). Other methods
may also be used to determine the same binding information. These
include, but are not limited to, ligand blot experiments using
labeled toxin, labeled glycosylated protein, or anti-toxin
antibodies, affinity chromatography, and in vitro binding of toxin
to intact BBMV.
5.18 Example 18
Construction of Plasmids with WT Cry3Bb Sequences
Standard recombinant DNA procedures were performed essentially as
described by Sambrook et al., (1989).
5.18.1 pEG1701
pEG1701 (FIG. 11), contained in EG11204 and EG11037, was
constructed by inserting the SphI-PstI fragment containing the
cry3Bb gene and the cry1F terminator from pEG911 (Baum, 1994) into
the SphI-PstI site of pEG854.9 (Baum et al., 1996), a high copy
number B. thuringiensis-E. coli shuttle vector.
5.18.2 pEG1028
pEG1028 contains the HindIII fragment of cry3Bb from pEG1701 cloned
into the multiple cloning site of pTZ18U at HindIII.
5.19 Example 19
Construction of Plasmids with Altered Cry3Bb Genes
Plasmid DNA from E. coli was prepared by the alkaline lysis method
(Maniatis et al., 1982) or by commercial plasmid preparation kits
(examples: PERFECTprep.TM. kit, 5 Prime-3 Prime, Inc., Bounder CO;
QIAGEN plasmid prep kit, QIAGEN Inc.). B. thuringiensis plasmids
were prepared from cultures grown in brain heart infusion plus 0.5%
glycerol (BHIG) to mid logarithmic phase by the alkaline lysis
method. When necessary for purification, DNA fragments were excised
from an agarose gel following electrophoresis and recovered by
glass milk using a Geneclean II.RTM. kit (BIO 101 Inc., La Jolla,
Calif.). Alteration of the cry3Bb gene was accomplished using
several techniques including site-directed mutagenesis, triplex
PCR.TM., quasi-random PCR.TM. mutagenesis, DNA shuffling and
standard recombinant techniques. These techniques are described in
Sections 6.1, 6.2, 6.3, 6.4 and 6.5, respectively. The DNA
sequences of primers used are listed in Section 7.
5.20 Example 20
Site-directed Mutagenesis
Site-directed mutagenesis was conducted by the protocols
established by Kunkle (1985) and Kunkle et al. (1987) using the
Muta-Gene.TM. M13 in vitro mutagenesis kit (Bio-Rad, Richmond,
Calif.). Combinations of alterations to cry3Bb were accomplished by
using the Muta-Gene.TM. kit and multiple mutagenic oligonucleotide
primers.
5.20.1 pEG1041
pEG1041, contained in EG11032, was constructed using the
Muta-Gene.TM. kit, primer C, and single-stranded pEG1028 as the DNA
template. The resulting altered cry3Bb DNA sequence was excised as
a PflMI DNA fragment and used to replace the corresponding DNA
fragment in pEG1701.
5.20.2 pEG1046
pEG1046, contained in EG11035, was constructed using the
Muta-Gene.TM. kit, primer D, and single-stranded pEG1028 as the DNA
template. The resulting altered cry3Bb DNA sequence was excised as
a PflMI DNA fragment and used to replace the corresponding DNA
fragment in pEG1701.
5.20.3 pEG1047
pEG1047, contained in EG11036, was constructed using the
Muta-Gene.TM. kit, primer E, and single-stranded pEG1028 as the DNA
template. The resulting altered cry3Bb DNA sequence was excised as
a PflMI DNA fragment and used to replace the corresponding DNA
fragment in pEG1701.
5.20.4 pEG1052
pEG1052, contained in EG11046, was constructed using the
Muta-Gene.TM. kit, primers D and E, and single-stranded pEG1028 as
the DNA template. The resulting altered cry3Bb DNA sequence was
excised as a PflMI DNA fragment and used to replace the
corresponding DNA fragment in pEG1701.
5.20.5 pEG1054
pEG1054, contained in EG11048, was constructed using the
Muta-Gene.TM. kit, primer F, and single-stranded pEG1028 as the DNA
template. The resulting altered cry3Bb DNA sequence was excised as
a PflMI DNA fragment and used to replace the corresponding DNA
fragment in pEG1701.
5.20.6 pEG1057
pEG1057, contained in EG1105 1, was constructed using the
Muta-Gene.TM. kit, primer G, and single-stranded pEG1028 as the DNA
template. The resulting altered cry3Bb DNA sequence was excised as
a PflMI DNA fragment and used to replace the corresponding DNA
fragment in pEG1701.
5.21 Example 21
Triplex PCR.TM.
Triplex PCR.TM. is described by Michael (1994). This method makes
use of a thermostable ligase to incorporate a phosphorylated
mutagenic primer into an amplified DNA fragment during PCR.TM..
PCR.TM. was performed on a Perkin Elmer Cetus DNA Thermal Cycler
(Perkin-Elmer, Norwalk, Conn.) using a AmpliTaq.TM. DNA polymerase
kit (Perkin-Elmer) and SphI-linearized pEG1701 as the template DNA.
PCR.TM. products were cleaned using commercial kits such as
Wizard.TM. PCR.TM. Preps (Promega, Madison, Wis.) and QIAquick
PCR.TM. Purification kit (QIAGEN Inc., Chatsworth, Calif.).
5.21.1 pEG1708 and pEG1709
pEG1708 and pEG1709, contained in EG11222 and EG11223,
respectively, were constructed by replacing the PflMI-PflMI
fragment of cry3Bb in pEG1701 with PflMI-digested and gel purified
PCR.TM. fragment altered at cry3Bb nucleotide positions 688-690,
encoding amino acid Y230. Random mutations were introduced into the
Y230 codon by triplex PCR.TM.. Mutagenic primer MVT095 was
phosphorylated and used together with outside primer pair FW001 and
FW006. Primer MVT095 also contains a silent mutation at position
687, changing T to C, which, upon incorporation, introduces an
additional EcoRI site into pEG1701.
5.21.2 pEG1710, pEG1711 and pEG1712
Plasmids pEG1701, pEG1711 and pEG1712, contained in EG11224,
EG11225 and EG11226, respectively, were created by replacing the
PflMI-PflMI fragment of the cry3Bb gene in pEG1701 with
PflMI-digested and gel purified PCR.TM. fragment altered at cry3Bb
nucleotide positions 690-692, encoding H231. Random mutations were
introduced into the H231 codon by triplex PCR.TM.. Mutagenic primer
MVT097 was phosphorylated and used together with outside primer
pair FW001 and FW006. Primer MVT097 also contains a T to C sequence
change at position 687 which, upon incorporation, results in an
additional EcoRI site by silent mutation.
5.21.3 pEG1713 and pEG1727
pEG1713 and pEG1727, contained in EG11227 and EG11242,
respectively, were constructed by replacing the PflMI-PflMI
fragment of the cry3Bb gene in pEG1701 with PflMI-digested and gel
purified PCR.TM. fragment altered at cry3Bb nucleotide positions
868-870, encoding amino acid R290. Triplex PCR.TM. was used to
introduce random changes into the R290 codon. The mutagenic primer,
MVT091, was designed so that the nucleotide substitutions would
result in approximately 36% of the sequences encoding amino acids D
or E. MVT091 was phosphorylated and used together with outside
primer pair FW001 and FW006.
5.22 Example 22
Quasi-random PCR.TM. Mutagenesis
Quasi-random mutagenesis combines the mutagenic PCR.TM. techniques
described by Vallette et al. (1989), Tomic et al. (1990) and LaBean
and Kauffman (1993). Mutagenic primers, sometimes over 70
nucleotides in length, were designed to introduce changes over
nucleotide positions encoding for an entire structural region, such
as a loop. Degenerate codons typically consisted of a ratio of 82%
WT nucleotide plus 6% each of the other 3 nucleotides per position
to semi-randomly introduce changes over the target region (LaBean
and Kauffman, 1993). When possible, natural restriction sites were
utilized; class 2s enzymes were used when natural sites were not
convenient (Stemmer and Morris, 1992, list additional restriction
enzymes useful to this technique). PCR.TM. was performed on a
Perkin Elmer Cetus DNA Thermal Cycler (Perkin-Elmer, Norwalk,
Conn.) using a AmpliTaq.TM. DNA polymerase kit (Perkin-Elmer) and
SphI-linearized pEG1701 as the template DNA. Quasi-random PCR.TM.
amplification was performed using the following conditions:
denaturation at 94.degree. C. for 1.5 min.; annealing at 50.degree.
C. for 2 min. and extension at 72.degree. C. for 3 min., for 30
cycles. The final 14 extension cycles were extended an additional
25 s per cycle. Primers concentration was 20 .mu.M per reaction or
40 .mu.M for long, mutagenic primers. PCR.TM. products were cleaned
using commercial kits such as Wizard.TM. PCR.TM. Preps (Promega,
Madison, Wis.) and QIAquick PCR.TM. Purification kit (QIAGEN Inc.,
Chatsworth, Calif.). In some instances PCR.TM. products were
treated with Klenow Fragment (Promega) following the manufacturer's
instructions to fill in any single base overhangs prior to
restriction digestion.
5.22.1 pEG1707
EG1707, contained in EG11221, was constructed by replacing the
PflMI-PflMI fragment of the cry3Bb gene in pEG1701 with
PflMI-digested and gel purified PCR.TM. fragment altered at cry3Bb
nucleotide positions 460-480, encoding 1.alpha.3,4 amino acids
154-160. Primer MVT075, which includes a recognition site for the
class 2s restriction enzyme BsaI, and primer FW006 were used to
introduce changes into this region by quasi-random mutagenesis.
Primers MVT076, also containing a BsaI site, and primer FW001 were
used to PCR.TM. amplify a "linker" fragment. Following PCR.TM.
amplification, both products were cleaned, end-filled, digested
with BsaI and ligated to each other. Ligated fragment was gel
purified and used as template for PCR.TM. amplification using
primer pair FW001 and FW006. PCR.TM. product was cleaned, digested
with PflMI, gel purified and ligated into PflMI-digested and
purified pEG1701 vector DNA.
5.22.2 pEG1720 and pEG1726
pEG1720 and pEG1726, contained in EG11234 and EG11241,
respectively, were constructed by replacing the PflMI-PflMI
fragment of the cry3Bb gene in pEG1701 with PflMI-digested and gel
purified PCR.TM. fragment altered at cry3Bb nucleotide positions
859-885, encoding 1.alpha.7,.beta.1 amino acids 287-295.
Quasi-random PCR.TM. mutagenesis was used to introduce changes into
this region. Mutagenic primer MVT111, designed with a BsaI site,
and primer FW006 were used to introduce the changes. Primer pair
MVT094, also containing a BsaI site, and FW001 were used to amplify
the linker fragment. The PCR.TM. products were digested with BsaI,
gel purified then ligated to each other. Ligated product was
PCR.TM. amplified using primer pair FW001 and FW006, digested with
PflMI.
5.22.3 pEG1714, pEG1715, pEG1716, pEG1718, pEG1719, pEG1722,
pEG1723, pEG1724 and pEG1725
pEG1714, pEG1715, pEG1716, pEG1718, pEG1719, pEG1722, pEG1723,
pEG1724 and pEG1725, contained in EG11228, EG11229, EG11230,
EG11232, EG11233, EG11236, EG11237, EG11238 and EG11239,
respectively, were constructed by replacing the PflMI-PflMI
fragment of the cry3Bb gene in pEG1701 with PflMI-digested and gel
purified PCR.TM. fragment altered at cry3Bb nucleotide positions
931-954, encoding 1.beta.1,.alpha.8 amino acids 311-318.
Quasi-random PCR.TM. mutagenesis was used to introduce changes into
this region using mutagenic primer MVT103 and primer FW006. Primers
FW001 and FW006 were used to amplify a linker fragment. The PCR.TM.
products were end-filled using Klenow and digested with BamHI. The
larger fragment from the FW001-FW006 digest was gel purified then
ligated to the digested MVT103-FW006 fragment. Ligated product was
gel purified and amplified by PCR.TM. using primer pair FW001 and
FW006. The amplified product was digested with PflMI and gel
purified prior to ligation into PflMI-digested and purified pEG1701
vector DNA.
5.22.4 pEG1701.1.beta.2.3
Plasmids carrying alterations of cry3Bb WT sequence at nucleotides
1051-1065, encoding structural region 1.beta.2,3 of Cry3Bb, were
constructed by replacing the MluI-SpeI fragment of pEG1701 with
isolated MluI- and SpeI-digested PCR.TM. product. The PCR.TM.
product was generated by quasi-random PCR.TM. mutagenesis were
mutagenic primer MVT081 was paired with FW006. These plasmids as a
group are designated pEG1701.1.beta.2,3.
5.22.5 pEG1701.1.beta.6,7
Plasmids containing mutations of the cry3Bb WT sequence at
nucleotides 1234-1248, encoding structural region 1.beta.6,7 of
Cry3Bb, were constructed by replacing the MluI-SpeI fragment of
pEG1701 with isolated MluI- and SpeI-digested PCR.TM. product. The
PCR.TM. product was generated by quasi-random PCR.TM. mutagenesis
where mutagenic primer MVT085 was paired with primer WD115. Primer
pair MVT089 and WD112 were used to amplify a linker fragment. Both
PCR.TM. products were digested with TaqI and ligated to each other.
The ligation product was gel purified and PCR.TM. amplified using
primer pair MVT089 and FW006. The amplified product was digested
with MluI and SpeI and ligated into MluI and SpeI digested and
purified pEG1701 vector DNA. These plasmids as a group are
designated pEG1701.1.beta.6,7.
5.22.6 pEG1701.110,11
Plasmids containing mutated cry3Bb sequences at nucleotides
1450-1467, encoding structural region 1.beta.10,11 of Cry3Bb, were
constructed by replacing the SpeI-PstI fragment of pEG1701 with
isolated SpeI- and PstI-digested PCR.TM. product. The PCR.TM.
product was generated by quasi-random PCR.TM. mutagenesis where
mutagenic primer MVT105 was paired with primer MVT070. Primer pair
MVT092 and MVT083 were used to generate a linker fragment. (MVT083
is a mutagenic oligo designed for another region. The sequence
changes introduced by MVT083 are removed following restriction
digestion and do not impact the alteration of cry3Bb in the
1.beta.10,11 region.) Both PCR.TM. products were digested with
BsaI, ligated together, and the ligation product PCR.TM. amplified
with primer pair MVT083 and MVT070. The resulting PCR.TM. product
was digested with SpeI and PstI, and gel purified. These plasmids
as a group are designated pEG1701.1.beta.10,11.
5.23 Example 23
DNA Shuffling
DNA-shuffling, as described by Stemmer (1994), was used to combine
individual alterations in the cry3Bb gene.
5.23.1 pEG1084, pEG1085, pEG1086 and pEG1087
pEG1084, pEG1085, pEG1086, and pEG1087, contained in EG11081,
EG11082, EG11083, and EG11084, respectively, were recovered from
DNA-shuffling. Briefly, PflMI DNA fragments were generated using
primer set A and B and each of the plasmids pEG1707, pEG1714,
pEG1715, pEG1716, pEG1041, pEG1046, pEG1047, and pEG1054 as DNA
templates. The resulting DNA fragments were pooled in equal-molar
amounts and digested with DNaseI and 50-100 bp DNA fragments were
recovered from an agarose gel by three successive freeze-thaw
cycles: three min in a dry-ice ethanol bath followed by complete
thawing at 50.degree. C. The recovered DNA fragments were assembled
by primerless-PCR.TM. and PCR.TM.-amplified using the primer set A
and B as described by Stemmer (1994). The final PCR.TM.-amplified
DNA fragments were cut with PflMI and used to replace the
corresponding cry3Bb PflMI DNA fragment in pEG1701.
5.24 Example 24
Recombinant DNA Techniques
Standard recombinant DNA procedures were performed essentially as
described by Sambrook et al. (1989).
5.24.1 pEG1717
pEG1717, contained in EG11231, was constructed by replacing the
small BglII fragment of pEG1710 with the small BglII fragment from
pEG1714.
5.24.2 pEG1721
pEG1721, contained in EG11235, was constructed by replacing the
small BglII fragment of pEG1710 with the small BglII fragment from
pEG1087.
5.24.3 pEG1063
pEG1062, contained in EG11057, was constructed by replacing the
NcoI DNA fragment containing ori 43 from pEG1054 with the isolated
NcoI DNA fragment containing ori 43 and the alterations in cry3Bb
from pEG1046.
5.24.4 pEG1063
pEG1063, contained in EG11058, was constructed by replacing the
NcoI DNA fragment containing ori 43 from pEG1054 with the isolated
NcoI DNA fragment containing ori 43 and the alterations in cry3Bb
from pEG1707.
5.24.5 pEG1095
pEG1095, contained in EG11095, was constructed by replacing the
MluI-SpeI DNA fragment in pEG1701 with the corresponding MluI-SpeI
DNA fragment from pEG1086.
5.25 Example 25
Primers Utilized in Constructing Cry3Bb* Variants
Shown below are the primers used for site-directed mutagenesis,
triplex PCR.TM. and quasi-random PCR.TM. to prepare the cry3Bb*
variants as described above. Primers were obtained from Ransom Hill
Bioscience, Inc. (Ramona, Calif.) and Integrated DNA Technologies,
Inc. (Coralville, Iowa). The specific composition of the primers
containing particular degeneracies at one or more residues is given
in Section 5.30, Example 30.
5.25.1 Primer FW001 (SEQ ID NO:71)
5'-AGACAACTCTACAGTAAAAGATG-3'
5.25.2 Primer FW006 (SEQ ID NO:72)
5'-GGTAATTGGTCAATAGAATC-3'
5.25.3 Primer MVT095 (SEQ ID NO:73)
5'-CAGAAGATGTTGCTGAATTCNNNCATAGACAATTAAAAC-3'
5.25.4 Primer MVT097 (SEQ ID NO:74)
5'-GATGTTGCTGAATTCJATNNNAGACAATTAAAAC-3'
5.25.5 Primer MVT091 (SEQ ID NO:75)
5'-CCCATTTTATGATATTBDNTTATACTCAAAAGG-3'
5.25.6 Primer MVT075 (SEQ ID NO:76)
5'-AGCTATGCTGGTCTCGGAAGAAAEFNFFNFJNJF
JFJNFINJFJAAAAGAAGCCAAGATCGAAT-3'
5.25.7 Primer MVT076 (SEQ ID NO:77)
5'-GGTCACCTAGGTCTCTCTTCCAGGAATTTAAC GCATTAAC-3'
5.25.8 Primer MVT111 (SEQ ID NO:78)
5'-AGCTATGCTGGTCTCCCAJTTJEHIEJEJJEIIKRR
JEHEIJEENIIIGTTAAAACAGAACTAAC-3'
5.25.9 Primer MVT094 (SEQ ID NO:79)
5'-ATCCAGTGGGGTCTCAAATGGGAAAAGTACA ATTAG-3'
5.25.10 Primer MVT103 (SEQ ID NO:80)
5'-CATTTTTACGGATCCAATTTTTJFFFJNEEJEFNFJ
NFEILEIJEOGGACCAACTTTTTTGAG-3'
5.25.11 Primer MVT081 (SEQ ID NO:81)
5'-GAATTTCATACGCGTCTTCAACCTGGTJEHJJJIIN
MEEIEJTCTTTCAATTATTGGTCTGG-3'
5.25.12 Primer MVT085 (SEQ ID NO:82)
5'-AAAAGTTTATCGAACFATAGCTAATACAGACGT
AGCGGCTJQQFFNEEJIIJEEIGTATATTTAGGTGT TACG-3'
5.25.13 Primer A (SEQ ID NO:83) 3b2pflm1
5'-GGAGTTCCATTTGCTGGGGC-3'
5.25.14 Primer B (SEQ ID NO:84) 3b2pflm2
5'-ATCTCCATAAAATGGGG-3'
5.25.15 Primer C (SEQ ID NO:85) 3b2165DG
5'-GCGAAGTAAAAGAAGCCAAGGTCGAATAAGGG-3'
5.25.16 Primer D (SEQ ID NO:86) 3b2160SKRD
5'-CCTTTAAGTTTGCGAAATCCACACAGCCAA GGTCGAATAAGGG-3'
5.25.17 Primer E (SEQ ID NO:87) 3b2290 VP
5'-CCCATTTTATGATGTTCGGTTATACCCAAAA GGGG-3'
5.25.18 Primer F (SEQ ID NO:88) 3b2EdA104
5'-GGCCAAGTGAAGACCCATGGAAGGC-3'
5.25.19 Primer G (SEQ ID NO:89) 3b2KG189
5'-GCAGTTTCCGGATTCGAAGTGC-3'
5.25.20 Primer WD112 (SEQ ID NO:90)
5'-CCGCTACGTCTGTATTA-3'
5.25.21 Primer WD115 (SEQ ID NO:91)
5'-ATAATGGAAGCACCTGA-3'
5.25.22 Primer MVT105 (SEQ ID NO:92)
5'-AGCTATGCTGGTCTCTTCTTAEJIFEIIEFFIJFIJIIN
ACAATTCCATTTTTTACTTGG-3'
5.25.23 Primer MVT092 (SEQ ID NO:93)
5'-ATCCAGTTGGGTCTCTAAGAAACAAACCGC GTAATTAAGC-3'
5.25.24 Primer MVT070 (SEQ ID NO:94)
5'-CCTCAAGGGTTATAACATCC-3'
5.25.25 Primer MVT083 (SEQ ID NO:95)
5'-GTACAAAAGCTAAGCTTTIEJIINPEEMEEIJN JESCGAACTATAGCTAATACAG-3'
5.26 Example 26
Sequence Analysis of Altered Cry3Bb Genes
E. coli DH5.alpha..TM. (GIBCO BRL, Gaithersburg, Md.), JM110 and
Sure.TM. (Stratagene, La Jolla, Calif.) cells were sometimes used
amplify plasmid DNA for sequencing. Plasmids were transformed into
these cells using the manufacturers' procedures. DNA was sequenced
using the Sequenase.RTM. 2.0 DNA sequencing kit purchased from U.S.
Biochemical Corporation (Cleveland, Ohio). The plasmids described
in Section 6, their respective diver-gence from WT cry3Bb sequence,
the resulting amino acid changes and the protein structure site of
the changes are listed in Table 11.
TABLE-US-00011 TABLE 11 DNA SEQUENCE CHANGES OF CRY3BB* GENES AND
RESULTING AMINO ACID SUBSTITUTIONS OF THE CRY3BB* PROTEINS
Structural Site of Plasmid cry3Bb* DNA Sequence Cry3Bb* Amino Acid
Sequence Alteration pEG1707 A460T, C461T, A462T, C464A, T465C,
T466C, T467A, T154F, P155H, L156H, L158R 1.alpha.3, 4 A468T, A469T,
G470C, T472C, T473G, G474T, A477T, A478T, G479C pEG1708 T687C,
T688C, A689T, C691A, A692G Y230L, H231S .alpha.6 pEG1709 T667C,
T687C, T688A, A689G, C691A, A692G S223P, Y230S .alpha.6 pEG1710
T687C, A692G H231R .alpha.6 pEG1711 T687C, C691A H231N, T241S
.alpha.6 pEG1712 T687C, C691A, A692C, T693C H231T .alpha.6 pEG1713
C868A, G869A, G870T H290N 1.alpha.7, .beta.1 pEG1714 C932T, A938C,
T942G, G949A, T954C S311L, N313T, E317K 1.beta.1, .alpha.8 pEG1715
T931A, A933C, T942A, T945A, G949A, A953G, S311T, E317K, Y318C
1.beta.1, .alpha.8 T954C pEG1716 T931G, A933C, C934G, T945G, C946T,
A947G, S311A, L312V, Q316W 1.beta.1, .alpha.8 G951A, T954C pEG1717
T687C, A692G, C932T, A938C, T942G, G949A, H231R, S311L, N313T,
E317K .alpha.6, 1.beta.1, .alpha.8 T954C pEG1718 T931A, A933G,
T935C, T936A, A938C, T939C, S311T, L312P, N313T, B317N 1.beta.1,
.alpha.8 T942C, T945A, G951T, T954C pEG1719 T931G, A933C, T936G,
T942C, C943T, T945A, S311A, Q316D 1.beta.1, .alpha.8 C946G, G948C,
T954C pEG1720 T861C, T866C, C868A, T871C, T872G, A875T, I289T,
L291R, Y292F, S293R 1.alpha.7, .beta.1 T877A, C878G, A882G pEG1721
T687C, A692G, C932T H231R, S311L .alpha.6, 1.beta.1, .alpha.8
pEG1722 T931A, C932T, A933C, T936C, T942G, T945A, T954C S311I
1.beta.1, .alpha.8 pEG1723 T931A, C932T, A933C, T936C, A937G,
A938T, S311I, N313H 1.beta.1, .alpha.8 C941A, T942C, T945A, C946A,
A947T, A950T, T954C pEG1724 A933C, T936C, A937G, A938T, C941A,
T942C, N313V, T314N, Q316M, E317V 1.beta.1, .alpha.8 T945A, C946A,
A947T, A950T, T954C pEG1725 A933T, A938G, T939G, T942A, T944C,
T945A, N313R, L315P, Q316L, E317A 1.beta.1, .alpha.8 A947T, G948T,
A950C, T954C pEG1726 A860T, T861C, G862A, C868T, G869T, T871C,
Y287F, D288N, R290L 1.alpha.7, .beta.1 A873T, T877A, C878G, A879T
pEG1727 C868G, G869T R290V 1.alpha.7, .beta.1 pE41041 A494G D165G
.alpha.4 pEG1046 G479A, A481C, A482C, A484C, G485A, S160N, K161P,
P162H R162H, .alpha.4 A486C, A494G D165G pEG1047 A865G, T877C
I289V, S293P 1.alpha.7, .beta.1 pEG1052 G479A, A481C, A482C, A484C,
G485A, A486C, S160N, K161P, P162H R162H, .alpha.4, 1.alpha.7,
.beta.1 A494G, A865G, D165G,T877C D165G, I289V, S293P pEG1054
T309A, .DELTA.310, .DELTA.311, .DELTA.312 D103E, .DELTA.A104
1.alpha.2a, 2b pEG1057 A565G, A566G K189G 1.alpha.4, 5 pEG1062
T309A, .DELTA.310, .DELTA.311, .DELTA.312, G479A, A481C, A482C,
D103E, .DELTA.A104, S160N, K161P, 1.alpha.2a, 2b .alpha.4 A484C,
G485A, A486C, A494G P162H R162H, D165G pEG1063 T309A, .DELTA.310,
.DELTA.311, .DELTA.312, A460T, C461T, A462T, D103E, .DELTA.A104,
T154F, P155H, 1.alpha.2, 2b 1.alpha.3, 4 C464A, T465C, T466C,
T467A, A468T, A469T, L156H, L158R G470C, T472C, T473G, G474T,
A477T, A478T, G479C pEG1084 A494G, T931A, A933C, T942A, T945A,
G949A, D165G, S311T, E317K .alpha.4, 1.beta.1, .alpha.8 T954C
pEG1085 A494G, A865G, T877C, T914C, T931G, A933C, D165G, I289V,
S293P, F305S, .alpha.4, 1.alpha.7, .beta.1 .beta.1, 1.beta.1,
.alpha.8 C934G, T945G, C946T, A947G, G951A, T954C, S311A, L312V,
Q316W, Q348R, .beta.2, .beta.3b A1043G, T1094C V365A pEG1086 A865G,
T877C, A1043G I289V, S293P, Q348R 1.alpha.7, .beta.1, .beta.2
pEG1087 A494G, C932T D165G, S311L .alpha.4, 1.beta.1, .alpha.8
pEG1095 A1043G Q348R .beta.2
5.27 Example 27
Expression of Cry3Bb* Proteins
5.27.1 Culture Conditions
LB agar was prepared using a standard formula (Maniatis et al.,
1982). Starch agar was obtained from Difco Laboratories (Detroit,
Mich.) and supplemented with an additional 5 g/l of agar. C2 liquid
medium is described by Donovan et al. (1988). C2 medium was
sometimes prepared without the phosphate buffer (C2-P). All
cultures were incubated at 25.degree. C. to 30.degree. C.; liquid
cultures were also shaken at 250 rpm, until sporulation and lysis
had occurred.
5.27.2 Transformation Conditions
pEG1701 and derivatives thereof were introduced into
acrystalliferious B. thuringiensis var. kurstaki EG7566 (Baum,
1994) or EG10368 (U.S. Pat. No. 5,322,687) by the electroporation
method of Macaluso and Mettus (1991). In some cases, the method was
modified as follows to maximize the number of transformants. The
recipient B. thuringiensis strain was inoculated from overnight
growth at 30.degree. C., on LB agar into brain heart infusion plus
0.5% glycerol, grown to an optical density of approximately 0.5 at
600 nm, chilled on ice for 10 min, washed 2.times. with EB and
resuspended in a 1/50 volume of EB. Transformed cells were selected
on LB agar or starch agar plus 5 .mu.g/ml chloramphenicol. Visual
screening of colonies was used to identify transformants producing
crystalline protein; those colonies were generally more opaque than
colonies that did not produce crystalline protein.
5.27.3 Strain and Protein Designations
A transformant containing an altered cry3Bb* gene encoding an
altered Cry3Bb* protein is designated by an "EG" number, e.g.,
EG11231. The altered Cry3Bb* protein is designated Cry3Bb followed
by the strain number, e.g., Cry3Bb.11231. Collections of proteins
with alterations at a structural site are designated Cry3Bb
followed by the structural site, e.g., Cry3Bb.1.beta.2,3. Table 12
lists the plasmids pertinent to this invention, the new B.
thuringiensis strains containing the plasmids, the acrystalliferous
B. thuringiensis recipient strain used, and the proteins produced
by the new strains.
5.28 Example 28
Generation and Characterization of Cry3Bb-60
5.28.1 Generation of Cry3Bb-60
Cry3Bb-producing strain EG7231 (U.S. Pat. No. 5,187,091) was grown
in C2 medium plus 3 mg/ml chloramphenicol. Following sporulation
and lysis, the culture was washed with water and Cry3Bb protein
purified by the NaBr solubilization and recrystallization method of
Cody et al. (1992). Protein concentration was determined by BCA
Protein Assay (Pierce, Rockford, Ill.). Recrystallized protein was
solubilized in 10 ml of 50 mM KOH per 100 mg of Cry3Bb protein and
buffered to pH 9.0 with 100 mM CAPS
(3-[cyclohexylamino]-1-propanesulfonic acid), pH 9.0. The soluble
toxin was treated with trypsin at a weight ratio of 50 mg toxin to
1 mg trypsin for 20 min to overnight at room temperature. Trypsin
cleaves proteins on the carboxyl side of available arginine and
lysine residues. For 8-dose bioassay, the solubilization conditions
were altered slightly to increase the concentration of protein: 50
mM KOH was added dropwise to 2.7 ml of a 12.77 mg/ml suspension of
purified Cry3Bb* until crystal solubilization occurred. The volume
was then adjusted to 7 ml with 100 mM CAPS, pH 9.0.
TABLE-US-00012 TABLE 12 PLASMIDS CARRYING ALTERED CRY3BB* GENES
TRANSFORMED INTO B. THURINGIENSIS FOR EXPRESSION OF ALTERED CRY3BB*
PROTEINS Plasmid Designation New BT Stain Expressed Protein pEG1701
EG11204 WT Cry3Bb pEG1701 EG11037 WT Cry3Bb PEG1707 EG11221
Cry3Bb.11221 pEG1708 EG11222 Cry3Bb.11222 pEG1709 EG11223
Cry3Bb.11223 pEG1710 EG11224 Cry3Bb.11224 pEG1711 EG11225
Cry3Bb.11225 pEG1712 EG11226 Cry3Bb.11226 pEG1713 EG11227
Cry3Bb.11227 pEG1714 EG11228 Cry3Bb.11228 pEG1715 EG11229
Cry3Bb.11229 pEG1716 EG11230 Cry3Bb.11230 pEG1717 EG11231
Cry3Bb.11231 pEG1718 EG11232 Cry3Bb.11232 pEG1719 EG11233
Cry3Bb.11233 pEG1720 EG11234 Cry3Bb.11234 pEG1721 EG11235
Cry3Bb.11235 pEG1722 EG11236 Cry3Bb.11236 pEG1723 EG11237
Cry3Bb.11237 pEG1724 EG11238 Cry3Bb.11238 pEG1725 EG11239
Cry3Bb.11239 pEG1726 EG11241 Cry3Bb.11241 pEG1727 EG11242
Cry3Bb.11242 pEG1041 EG11032 Cry3Bb.11012 pEG1046 EG11035
Cry3Bb.11035 pEG1047 EG11036 Cry3Bb.11036 pEG1052 EG11046
Cry3Bb.11046 pEG1054 EG11048 Cry3Bb.11048 pEG1057 EG11051
Cry3Bb.11051 pEG1062 EG11057 Cry3Bb.11057 pEG1063 EG11058
Cry3Bb.11058 pEG1084 EG11081 Cry3Bb.11081 pEG1085 EG11082
Cry3Bb.11082 pEG1086 EG11083 Cry3Bb.11083 pEG1087 EG11084
Cry3Bb.11084 pEG1095 EG11095 Cry3Bb.11095 pEG1098 EG11098
Cry3Bb.11098 pEG1701.1.beta.2,3 collection of unnamed strains
Cry3Bb.1.beta.2,3 pEG1701.1.beta.6,7 collection of unnamed strains
Cry3Bb.1.beta.6,7 pEG1701.1.beta.10,11 collection of unnamed
strains Cry3Bb.1.beta.10,11
5.28.2 Determination of Molecular Weight of Cry3Bb-60
The molecular weight of the predominant trypsin digestion fragment
of Cry3Bb was determined to be 60 kDa by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) analysis using commercial molecular
weight markers. This digestion fragment is designated Cry3Bb-60. No
further digestion of the 60 kDa cleavage product was observed.
5.28.3 Determination of NH.sub.2-terminus of Cry3Bb-60
To determine the NH.sub.2-terminal sequence of Cry3Bb-60, the
trypsin digest was fractionated by SDS-PAGE and transferred to
Immobilon.TM.-P membrane (Millipore Corporation, Bedford, Mass.)
following standard western blotting procedures. After transfer, the
membrane was rinsed twice with water then stained with 0.025%
Coomassie Brilliant Blue R-250 plus 40% methanol for 5 min,
destained with 50% methanol and rinsed in water. The Cry3Bb.60 band
was excised with a razor blade. NH.sub.2-terminal sequencing was
performed at the Tufts Medical School, Department of Physiology
(Boston, Mass.) using standard automated Edman degradation
procedures. The NH.sub.2-terminal amino acid sequence was
determined to be SKRSQDR (SEQ ID NO:96), corresponding to amino
acids 160-166 of Cry3Bb. Trypsin digestion occurred on the carboxyl
side of amino acid R159 resulting in the removal of helices
1-3.
5.29 Example 29
Bioactivity of Cry3Bb* Proteins
5.29.1 Culture Conditions and Protein Concentration
Determination
Cultures for 1-dose bioassays were grown in C2-P plus 5 .mu.g/ml
chloramphenicol (C2-P/cm5) then diluted with 3 volumes of 0.005%
Triton X-100.RTM.. The protein concentrations of these cultures
were not determined. Cultures for 8-dose bioassays were grown in
C2/cm5, washed 1-2 times with 1-2 volumes of sterile water and
resuspended in 1/10 volume of sterile 0.005% Triton X-100.RTM.. The
toxin protein concentration of each concentrate was determined as
described by Brussock and Currier (1990), omitting the treatment
with 3 M HEPES. The protein concentration was adjusted to 3.2 mg/ml
in 0.005% Triton X-100.RTM. for the top dose of the assay.
Cry3Bb.60 was produced and quantified for 8-dose assay as described
in Section 9.1.
5.29.2 Insect Bioassays
Diabrotica undecimpunctata howardi Barber (southern corn rootworm
or SCRW) and Diabrotica virgifera virgifiera LeConte (western corn
rootworm or WCRW) larvae were reared as described by Slaney et al.
(1992). Eight-dose assays and probit analyses were performed as
described by Slaney et al. (1992). Thirty-two larvae were tested
per dose at 50 .mu.l of sample per well of diet (surface area of
175 mm.sup.2). Positive controls were WT Cry3Bb-producing strains
EG11037 or EG11204. All bioassays were performed using 128-well
trays containing approximately 1 ml of diet per well with
perforated mylar sheet covers (C-D International Inc., Pitman,
N.J.). One-dose assays were performed essentially the same except
only 1 dose was tested per strain. All assay were replicated at
least twice.
5.29.3 Insect Bioassay Results: 1-Dose Assays Against SCRW
Results from 1-dose assays are expressed as the relative mortality
(RM) of the experimental strain compared to WT (% mortality of
experimental culture divided by % mortality of WT culture). Altered
and improved Cry3Bb proteins derived from plasmids constructed
using PCR.TM. methods introducing random or semi-random changes
into the cry3Bb gene sequence were distinguished from other altered
but not improved Cry3Bb proteins by replicated, 1-dose assay
against SCRW larvae. Those proteins showing increased activity
(defined as RM.gtoreq.1.5) compared to WT Cry3Bb or, in the case of
proteins with combinations of altered sites, compared to a
"parental" altered Cry3Bb protein were further characterized by
8-dose assay. The overall RM "pattern" produced by 1-dose assay
results from a collection of proteins carrying random or
semi-random alterations within a single structural region, e.g., in
1.beta.2,3, can be used to determine if that structural region is
important for bioactivity. Retention of WT levels of activity
(RM.apprxeq.1) indicate changes are tolerated in that region.
Overall loss of activity (RM<1) distinguishes the region as
important for bioactivity.
5.29.4 Cry3Bb.1.beta.2,3: Results of 1-Dose Bioassays Against
SCRW
Cry3Bb.1.beta.2.3 protein are a collection of proteins altered in
the 1.beta.2,3 region of Cry3Bb (see Section 5.3.4). Typical
results of 1-dose assays of these altered proteins are shown in
FIG. 12. The RM values for Cry3Bb.1.beta.2,3 proteins are less than
1, with a few exceptions of values close to 1, indicating that this
region is important for toxicity.
5.29.5 Cry3Bb.1.beta.6,7: Results of 1-Dose Bioassays Against
SCRW
Cry3Bb.1.beta.6,7 proteins are a collection of proteins altered in
the 1.beta.6,7 region of Cry3Bb (see Section 5.3.5). Typical
results of 1-dose assays of these altered proteins are shown in
FIG. 13. With a few exceptions of values close to 1, the RM values
for Cry3Bb.1.beta.6,7 proteins are less than 1, indicating that
this region is important for toxicity.
5.29.6 Cry3Bb.1.beta.10,11: Results of 1-Dose Bioassays Against
SCRW
Cry3Bb.1.beta.10,11 proteins are a collection of proteins altered
in the 1.beta.10,11 region of Cry3Bb (see Section 5.3.6). Typical
results of 1-dose assays of these altered proteins are shown in
FIG. 14. With a few exceptions of values close to 1, the RM values
for Cry3Bb.1.beta.10,11 proteins are less than 1, indicating that
this region is important for bioactivity.
5.29.7 Insect Bioassay Results: Results of 8-Dose Assays Against
SCRW
Results from 8-dose assays are expressed as an LC.sub.50 value
(protein concentration giving 50% mortality) with 95% confidence
intervals. The LC.sub.50 values with 95% confidence intervals of
altered Cry3Bb proteins showing improved activities against SCRW
larvae and LC.sub.50 values of the WT Cry3Bb control determined at
the same time are listed in Table 13 along with the fold increase
over WT activity for each improved protein.
TABLE-US-00013 TABLE 13 DESIGNED CRY.3BB PROTEINS WERE TESTED
AGAINST SCREW LARVAE IN REPLICATED, 8-DOSE ASSAYS TO DETERMINE THE
IC.sub.50 VALUES IC.sub.50 .mu.g/well (95% C.I.) Fold In- WT Cry3Bb
crease Over Improved Protein Improved Protein Control WT Activity
Cry3Bb.60 6.7 (5.3-8.4) 24.1 (15-39) 3.6x Cry3Bb.11221 3.2 (2.5-4)
20.5 (14.5-29) 6.4x Cry3Bb.11222 7.3 (6-9) 29.4 (23-37) 4.0x
Cry3Bb.11223 19.5 (9-12) 29.4 (23-37) 2.8x Cry3Bb.11224 6.5
(5.1-8.2) 32.5 (25-43) 5.0x Cry3Bb.11225 13.7 (11-16.8) 49.5
(39-65) 3.6x Cry3Bb.11226 16.7 (10.6-24.2) 49.5 (39-65) 3.0x
Cry3Bb.11227 11.1 (9.1-13.5) 21.3 (16-28) 1.9x Cry3Bb.11228 8.0
(6.6-9.8) 32.9 (25-45) 4.1x Cry3Bb.11229 7.2 (5.8-8.8) 18.2 (15-22)
2.5x Cry3Bb.11230 7.0 (5.8-8.6) 32.9 (25-45) 4.7x Cry3Bb.11231 3.3
(3.0-3.7) 26.1 (22-31) 7.9x Cry3Bb.11232 6.4 (5.4-7.7) 32.9 (25-45)
5.1x Cry3Bb.11233 15.7 (12-20) 32.9 (25-45) 2.2x Cry3Bb.11234 7
(6-9) 29 (22-39) 4.1x Cry3Bb.11235 4.2 (3.6-4.9) 13.3 (10-17) 3.2x
Cry3Bb.11236 11.6 (9-15) 36.4 (27-49) 3.1x Cry3Bb.11237 6.8 (4-11)
36.4 (27-49) 5.4x Cry3Bb.11238 13.9 (11-17) 36.4 (27-49) 2.6x
Cry3Bb.11239 13.0 (10-16) 36.4 (27-49) 2.8x Cry3Bb.11241 11 (7-16)
29 (22-39) 2.6x Cry3Bb.11242 11.9 (9.2-16) 30 (23-38) 2.5x
Cry38b.11032 4.2 (3.6-4.9) 13.3 (10-17) 3.1x Cry3Bb.11035 10.3
(8-13) 27.9 (23-34) 2.7x Cry3Bb.11036 6.5 (5.1-7.9) 27.9 (23-34)
4.3x Cry3Bb.11046 12.1 (8-19) 31.2 (25-39) 2.6x Cry3Bb.11048 8.3
(6-11) 35.4 (24-53) 4.3x Cry3Bb.11051 11.8 (8-16) 35.4 (24-53) 3.0x
Cry3Bb.11057 8.8 (7-11) 29.5 (24-36) 3.4x Cry3Bb.11058 9.6 (6-14)
33.4 (27-43) 3.5x Cry3Bb.11081 8.5 (7-11) 51.5 (37-79) 6.1x
Cry3Bb.11082 10.6 (8-13) 51.5 (37-79) 4.9x Cry3Bb.11083 7.0 (5-10)
51.5 (37-79) 7.4x Cry3Bb.11084 7.2 (4-12) 51.5 (37-79) 7.2x
Cry3Bb.11095 11.1 (9-14) 51.5 (37-79) 4.6x Cry3Bb.11098
5.29.8 Insect Bioassay Results: 8-Dose Assays Against WCRW
WCRW larvae are delicate and difficult to work with. Therefore,
only some of the designed Cry3Bb showing improved activity against
SCRW larvae were also tested against WCRW larvae in 8-dose assays.
The LC.sub.50 determinations for the designed Cry3Bb proteins are
shown in Table 14 along with the LC.sub.50 values of the WT Cry3Bb
control determined at the same time.
TABLE-US-00014 TABLE 14 CRY3BB* PROTEINS SHOWING IMPROVED ACTIVITY
AGAINST SCRW LARVAE ALSO SHOW IMPROVED ACTIVITY AGAINST WCRW LARVAE
IC.sub.50 .mu.g/well (95% C.I.) Fold Increase WT Cry3Bb Over
Improved Protein Improved Protein Control WT Activity EG11083 6.3
(4.7-8.2) 63.5 (46-91) 10.1x EG11230 4.5 (2.1-7.4) 24.2 (13-40)
5.4x EG11231 2.5 (1.7-3.6) 32.2 (14-67) 12.9x
5.30 Example 30
Channel Activity
Ion channels produced by Cry3Bb and some of its derivatives were
measured by the methods described by Slatin et al. (1990). In some
instances, lipid bilayers were prepared from a mixture of 4:1
phophatidylethanolamine (PE): phosphatidylcholine (PC). Toxin
protein was solubilized from washed, C2 medium, B. thuringiensis
cultures with 12 mM KOH. Following centrifugation to remove spores
and other debris, 10 .mu.g of soluble toxin protein was added to
the cis compartment (4.5 ml volume) of the membrane chamber.
Protein concentration was determined using the BCA Protein Assay
(Pierce).
5.30.1 Channel Activity of WT Cry3Bb.
Upon exposure to black lipid membranes, Cry3Bb forms ion channels
with various conductance states. The channels formed by Cry3Bb are
rarely discrete channels with well resolved open and closed states
and usually require incubation of the toxin with the membrane for
30-45 min before any channel-like events are observed. After
formation of the initial conductances, the size increases from
approximately 200 pS to over 10,000 pS over 2-3 h. Only the small
conductances (.ltoreq.200 pS) are voltage dependent. Over 200 pS,
the conductances are completely symmetric. Cry3Bb channels also
exhibit .beta.-mercaptoethanol-dependent activation, growing from
small channel conductances of .about.200 pS to several thousand pS
within 2 min of the addition of .beta.-mercaptoethanol to the cis
compartment of the membrane chamber.
5.30.2 Cry3Bb.11032
The channel activity of Cry3Bb.11032 is much like WT Cry3Bb when
the solubilized toxin protein is added to the cis compartment of
the membrane chamber. However, when this protein is artificially
incorporated into the membrane by forming or "painting" the
membrane in the presence of the Cry3Bb.11032 protein, a 16-fold
increase in the initial channel conductances is observed
(.about.4000 pS). This phenomenon is not observed with WT
Cry3Bb.
5.30.3 Cry3Bb.11035
Upon exposure to artificial membranes, the Cry3Bb.11035 protein
spontaneously forms channels that grow to large conductances within
a relatively short time span (.about.5 min). Conductance values
ranges from 3000-6000 pS and, like WT Cry3Bb, are voltage dependent
at low conductance values.
5.30.4 Cry3Bb.11048
The Cry3Bb.11048 protein is quite different than WT Cry3Bb in that
it appears not to form channels at all, but, rather, forms
symmetrical pores with respect to voltage. Once the pore is formed,
it remains open and allows a steady conductance ranging from 25 to
130 pS.
5.30.5 Cry3Bb.11224 and Cry3Bb.11226
The metal binding site of WT Cry3Bb formed by H231 in the dimer
structure was removed in proteins Cry3Bb.11224 and Cry3Bb.11226.
The conductances formed by both designed proteins are identical to
that of WT Cry3Bb with the exception that neither of the designed
proteins exhibits .beta.-mercaptoethanol-dependent activation.
5.30.6 Cry3Bb.11221
Cry3Bb.11221 protein has been observed to immediately form small
channels of 100-200 pS with limited voltage dependence. Some higher
conductances were observed at the negative potential. In other
studies, the onset of activity was delayed by 27 min, which is more
typical for WT Cry3Bb. Unlike WT Cry3Bb, however, Cry3Bb.11221
forms well resolved, 600 pS channels with long open states. The
protein eventually reaches conductances of 7000 pS.
5.30.7 Cry3Bb.11242
Cry3Bb.11242 protein forms small conductances immediately upon
exposure to an artificial membrane. The conductances grow steadily
and rapidly to 6000 pS in approximately 3 min. Some voltage
dependence was noted with a preference for a negative imposed
voltage.
5.30.8 Cry3Bb.11230
Unlike WT Cry3Bb, Cry3Bb.11230 forms well resolved channels with
long open states that do not continue to grow in conductance with
time. The maximum observed channel conductances reached 3000 pS.
FIG. 15 illustrates the difference between the channels formed by
Cry3Bb and Cry3Bb.11230.
5.30.9 Cry3Bb.60
Cry3Bb.60 forms well resolved ion channels within 20 min of
exposure to an artificial membrane. These channels grow in
conductance and frequency with time. The behavior of Cry3Bb.60 in a
planar lipid bilayer differs from Cry3Bb in two significant ways.
The conductances created by Cry3Bb.60 form more quickly than Cry3Bb
and, unlike Cry3Bb, the conductances are stable, having well
resolved open and closed states definitive of stable ion channels
(FIG. 16).
TABLE-US-00015 TABLE 15 SEQ ID NO:83 % of Nucleotide in mixture
Code A T G C N 25 25 25 25
TABLE-US-00016 TABLE 16 SEQ ID NO:84 % of Nucleotide in mixture
Code A T G C N 25 25 25 25
TABLE-US-00017 TABLE 17 SEQ ID NO:85 % of Nucleotide in mixture
Code A T G C B 16 16 52 16 D 70 10 10 10 N 25 25 25 25
TABLE-US-00018 TABLE 18 SEQ ID NO:86 % of Nucleotide in mixture
Code A T G C E 82 6 6 6 F 6 6 6 82 J 6 82 6 6 I 6 6 82 6 N 25 25 25
25
TABLE-US-00019 TABLE 19 SEQ ID NO:88 % of Nucleotide in mixture
Code A T G C J 6 82 6 6 E 82 6 6 6 H 1 1 1 97 I 6 6 82 6 K 15 15 15
55 R 15 55 15 15
TABLE-US-00020 TABLE 20 SEQ ID NO:90 % of Nucleotide in mixture
Code A T G C J 6 82 6 6 F 6 6 6 82 N 25 25 25 25 E 82 6 6 6 I 6 6
82 6 L 8 1 83 8 O 1 1 1 97
TABLE-US-00021 TABLE 21 SEQ ID NO:91 % of Nucleotide in mixture
Code A T G C J 6 82 6 6 E 82 6 6 6 H 1 1 1 97 I 6 6 82 6 N 25 25 25
25 M 82 2 8 8
TABLE-US-00022 TABLE 22 SEQ ID NO:92 % of Nucleotide in mixture
Code A T G C J 6 82 6 6 Q 0 9 82 9 F 6 6 6 82 N 25 25 25 25 E 82 6
6 6 I 6 6 82 6
TABLE-US-00023 TABLE 23 SEQ ID NO:92 % of Nucleotide in mixture
Code A T G C J 6 82 6 6 F 6 6 6 82 N 25 25 25 25 E 82 6 6 6 I 6 6
82 6
TABLE-US-00024 TABLE 24 SEQ ID NO:95 % of Nucleotide in mixture
Code A T G C J 6 82 6 6 N 25 25 25 25 E 82 6 6 6 I 6 6 82 6 M 82 2
8 8 P 8 2 8 82 S 1 97 1 1
5.32 Example 32
Atomic Coordinates for Cry3Bb
The atomic coordinates of the Cry3Bb protein are given in the
Appendix included in Section 9.1
5.33 Example 33
Atomic Coordinates for Cry3A
The atomic coordinates of the Cry3A protein are given in the
Appendix included in Section 9.2
5.34 Example 34
Modification of Cry Genes for Expression in Plants
Wild-type cry genes are known to be expressed poorly in plants as a
full length gene or as a truncated gene. Typically, the G+C content
of a cry gene is low (37%) and often contains many A+T rich
regions, potential polyadenylation sites and numerous ATTTA
sequences. Table 25 shows a list of potential polyadenylation
sequences which should be avoided when preparing the "plantized"
gene construct.
TABLE-US-00025 TABLE 25 LIST OF SEQUENCES OF THE POTENTIAL
POLYADENYLATION SIGNALS AATAAA* AAGCAT AATAAT* AATAAT AACCAA ATACAT
ATATAA AAAATA AATCAA ATTAAA** ATACTA AATTAA** ATAAAA AATACA**
ATGAAA CATAAA** *indicates a potential major plant polyadenylation
site. **indicates a potential minor animal polyadenylation site.
All others are potential minor plant polyadenylation sites.
The regions for mutagenesis may be selected in the following
manner. All regions of the DNA sequence of the cry gene are
identified which contained five or more consecutive base pairs
which were A or T. These were ranked in terms of length and highest
percentage of A+T in the surrounding sequence over a 20-30 base
pair region. The DNA is analysed for regions which might contain
polyadenylation sites or ATTTA sequences. Oligonucleotides are then
designed which maximize the elimination of A+T consecutive regions
which contained one or more polyadenylation sites or ATTTA
sequences. Two potential plant polyadenylation sites have been
shown to be more critical based on published reports. Codons are
selected which increase G+C content, but do not generate
restriction sites for enzymes useful for cloning and assembly of
the modified gene (e.g. BamHI, BglII, SacI, NcoI, EcoRV, etc.).
Likewise condons are avoided which contain the doublets TA or GC
which have been reported to be infrequently-found codons in
plants.
Although the CaMV35S promoter is generally a high level
constitutive promoter in most plant tissues, the expression level
of genes driven the CaMV35S promoter is low in floral tissue
relative to the levels seen in leaf tissue. Because the
economically important targets damaged by some insects are the
floral parts or derived from floral parts (e.g., cotton squares and
bolls, tobacco buds, tomato buds and fruit), it is often
advantageous to increase the expression of crystal proteins in
these tissues over that obtained with the CaMV35S promoter.
The 35S promoter of Figwort Mosaic Virus (FMV) is analogous to the
CaMV35S promoter. This promoter has been isolated and engineered
into a plant transformation vector. Relative to the CaMV promoter,
the FMV 35S promoter is highly expressed in the floral tissue,
while still providing similar high levels of gene expression in
other tissues such as leaf. A plant transformation vector, may be
constructed in which the full length synthetic cry gene is driven
by the FMV 35S promoter. Tobacco plants may be transformed with the
vector and compared for expression of the crystal protein by
Western blot or ELISA immunoassay in leaf and floral tissue. The
FMV promoter has been used to produce relatively high levels of
crystal protein in floral tissue compared to the CaMV promoter.
5.35 Example 35
Expression of Synthetic Cry Genes with ssRUBISCO Promoters and
Chloroplast Transit Peptides
The genes in plants encoding the small subunit of RUBISCO (SSU) are
often highly expressed, light regulated and sometimes show tissue
specificity. These expression properties are largely due to the
promoter sequences of these genes. It has been possible to use SSU
promoters to express heterologous genes in transformed plants.
Typically a plant will contain multiple SSU genes, and the
expression levels and tissue specificity of different SSU genes
will be different. The SSU proteins are encoded in the nucleus and
synthesized in the cytoplasm as precursors that contain an
N-terminal extension known as the chloroplast transmit peptide
(CTP). The CTP directs the precursor to the chloroplast and
promotes the uptake of the SSU protein into the chloroplast. In
this process, the CTP is cleaved from the SSU protein. These CTP
sequences have been used to direct heterologous proteins into
chloroplasts of transformed plants.
The SSU promoters might have several advantages for expression of
heterologous genes in plants. Some SSU promoters are very highly
expressed and could give rise to expression levels as high or
higher than those observed with the CaMV35S promoter. The tissue
distribution of expression from SSU promoters is different from
that of the CaMV35S promoter, so for control of some insect pests,
it may be advantageous to direct the expression of crystal proteins
to those cells in which SSU is most highly expressed. For example,
although relatively constitutive, in the leaf the CaMV35S promoter
is more highly expressed in vascular tissue than in some other
parts of the leaf, while most SSU promoters are most highly
expressed in the mesophyll cells of the leaf. Some SSU promoters
also are more highly tissue specific, so it could be possible to
utilize a specific SSU promoter to express the protein of the
present invention in only a subset of plant tissues, if for example
expression of such a protein in certain cells was found to be
deleterious to those cells. For example, for control of Colorado
potato beetle in potato, it may be advantageous to use SSU
promoters to direct crystal protein expression to the leaves but
not to the edible tubers.
Utilizing SSU CTP sequences to localize crystal proteins to the
chloroplast might also be advantageous. Localization of the B.
thuringiensis crystal proteins to the chloroplast could protect
these from proteases found in the cytoplasm. This could stabilize
the proteins and lead to higher levels of accumulation of active
toxin, cry genes containing the CTP could be used in combination
with the SSU promoter or with other promoters such as CaMV35S.
5.36 Example 36
Targeting of Cry* Proteins to the Extracellular Space or Cacuole
through the Use of Signal Peptides
The B. thuringiensis proteins produced from the synthetic genes
described here are localized to the cytoplasm of the plant cell,
and this cytoplasmic localization results in plants that are
insecticidally effective. It may be advantageous for some purposes
to direct the B. thuringiensis proteins to other compartments of
the plant cell. Localizing B. thuringiensis proteins in
compartments other than the cytoplasm may result in less exposure
of the B. thuringiensis proteins to cytoplasmic proteases leading
to greater accumulation of the protein yielding enhanced
insecticidal activity. Extracellular localization could lead to
more efficient exposure of certain insects to the B. thuringiensis
proteins leading to greater efficacy. If a B. thuringiensis protein
were found to be deleterious to plant cell function, then
localization to a noncytoplasmic compartment could protect these
cells from the protein.
In plants as well as other eukaryotes, proteins that are destined
to be localized either extracellularly or in several specific
compartments are typically synthesized with an N-terminal amino
acid extension known as the signal peptide. The signal peptide
directs the protein to enter the compartmentalization pathway, and
it is typically cleaved from the mature protein as an early step in
compartmentalization. For an extracellular protein, the secretory
pathway typically involves cotranslational insertion into the
endoplasmic reticulum with cleavage of the signal peptide occurring
at this stage. The mature protein then passes through the Golgi
body into vesicles that fuse with the plasma membrane thus
releasing the protein into the extracellular space. Proteins
destined for other compartments follow a similar pathway. For
example, proteins that are destined for the endoplasmic reticulum
or the Golgi body follow this scheme, but they are specifically
retained in the appropriate compartment. In plants, some proteins
are also targeted to the vacuole, another membrane bound
compartment in the cytoplasm of many plant cells. Vacuole targeted
proteins diverge from the above pathway at the Golgi body where
they enter vesicles that fuse with the vacuole.
A common feature of this protein targeting is the signal peptide
that initiates the compartmentalization process. Fusing a signal
peptide to a protein will in many cases lead to the targeting of
that protein to the endoplasmic reticulum. The efficiency of this
step may depend on the sequence of the mature protein itself as
well. The signals that direct a protein to a specific compartment
rather than to the extracellular space are not as clearly defined.
It appears that many of the signals that direct the protein to
specific compartments are contained within the amino acid sequence
of the mature protein. This has been shown for some vacuole
targeted proteins, but it is not yet possible to define these
sequences precisely. It appears that secretion into the
extracellular space is the "default" pathway for a protein that
contains a signal sequence but no other compartmentalization
signals. Thus, a strategy to direct B. thuringiensis proteins out
of the cytoplasm is to fuse the genes for synthetic B.
thuringiensis genes to DNA sequences encoding known plant signal
peptides. These fusion genes will give rise to B. thuringiensis
proteins that enter the secretory pathway, and lead to
extracellular secretion or targeting to the vacuole or other
compartments. Signal sequences for several plant genes have been
described. One such sequence is for the tobacco pathogenesis
related protein PR1b has been previously described (Comelissen et
al, 1986). The PR1b protein is normally localized to the
extracellular space. Another type of signal peptide is contained on
seed storage proteins of legumes. These proteins are localized to
the protein body of seeds, which is a vacuole like compartment
found in seeds. A signal peptide DNA sequence for the
.beta.-subunit of the 7S storage protein of common bean (Phaseolus
vulgaris), PvuB has been described (Doyle et al, 1986). Based on
the published these published sequences, genes may be synthesized
chemically using oligonucleotides that encode the signal peptides
for PR1b and PvuB. In some cases to achieve secretion or
compartmentalization of heterologous proteins, it may be necessary
to include some amino acid sequence beyond the normal cleavage site
of the signal peptide. This may be necessary to insure proper
cleavage of the signal peptide.
5.37 Example 37
Isolation of Transgenic Maize Resistant to Diabrotica spp. Using
Cry3Bb Variants
5.37.1 Plant Gene Construction
The expression of a plant gene which exists in double-stranded DNA
form involves transcription of messenger RNA (mRNA) from one strand
of the DNA by RNA polymerase enzyme, and the subsequent processing
of the mRNA primary transcript inside the nucleus. This processing
involves a 3' non-translated region which adds polyadenylate
nucleotides to the 3' end of the RNA. Transcription of DNA into
mRNA is regulated by a region of DNA usually referred to as the
"promoter". The promoter region contains a sequence of bases that
signals RNA polymerase to associate with the DNA and to initiate
the transcription of mRNA using one of the DNA strands as a
template to make a corresponding strand of RNA.
A number of promoters which are active in plant cells have been
described in the literature. Such promoters may be obtained from
plants or plant viruses and include, but are not limited to, the
nopaline synthase (NOS) and octopine synthase (OCS) promoters
(which are carried on tumor-inducing plasmids of Agrobacterium
tumefaciens), the cauliflower mosaic virus (CaMV) 19S and 35S
promoters, the light-inducible promoter from the small subunit of
ribulose 1,5-bisphosphate carboxylase (ssRUBISCO, a very abundant
plant polypeptide), and the Figwort Mosaic Virus (FMV) 35S
promoter. All of these promoters have been used to create various
types of DNA constructs which have been expressed in plants (see
e.g., U.S. Pat. No. 5,463,175, specifically incorporated herein by
reference).
The particular promoter selected should be capable of causing
sufficient expression of the enzyme coding sequence to result in
the production of an effective amount of protein. One set of
preferred promoters are constitutive promoters such as the CaMV35S
or FMV35S promoters that yield high levels of expression in most
plant organs (U.S. Pat. No 5,378,619, specifically incorporated
herein by reference). Another set of preferred promoters are root
enhanced or specific promoters such as the CaMV derived 4 as-1
promoter or the wheat POX1 promoter (U.S. Pat. No. 5,023,179,
specifically incorporated herein by reference; Hertig et al.,
1991). The root enhanced or specific promoters would be
particularly preferred for the control of corn rootworm
(Diabroticus spp.) in transgenic corn plants.
The promoters used in the DNA constructs (i.e. chimeric plant
genes) of the present invention may be modified, if desired, to
affect their control characteristics. For example, the CaMV35S
promoter may be ligated to the portion of the ssRUBISCO gene that
represses the expression of ssRUBISCO in the absence of light, to
create a promoter which is active in leaves but not in roots. The
resulting chimeric promoter may be used as described herein. For
purposes of this description, the phrase "CaMV35S" promoter thus
includes variations of CaMV35S promoter, e.g., promoters derived by
means of ligation with operator regions, random or controlled
mutagenesis, etc. Furthermore, the promoters may be altered to
contain multiple "enhancer sequences" to assist in elevating gene
expression.
The RNA produced by a DNA construct of the present invention also
contains a 5' non-translated leader sequence. This sequence can be
derived from the promoter selected to express the gene, and can be
specifically modified so as to increase translation of the mRNA.
The 5' non-translated regions can also be obtained from viral
RNA's, from suitable eucaryotic genes, or from a synthetic gene
sequence. The present invention is not limited to constructs
wherein the non-translated region is derived from the 5'
non-translated sequence that accompanies the promoter sequence.
For optimized expression in monocotyledenous plants such as maize,
an intron should also be included in the DNA expression construct.
This intron would typically be placed near the 5' end of the mRNA
in untranslated sequence. This intron could be obtained from, but
not limited to, a set of introns consisting of the maize hsp70
intron (U.S. Pat. No. 5,424,412; specifically incorporated herein
by reference) or the rice Act1 intron (McElroy et al., 1990). As
shown below, the maize hsp70 intron is useful in the present
invention.
As noted above, the 3' non-translated region of the chimeric plant
genes of the present invention contains a polyadenylation signal
which functions in plants to cause the addition of adenylate
nucleotides to the 3' end of the RNA. Examples of preferred 3'
regions are (1) the 3' transcribed, non-translated regions
containing the polyadenylate signal of Agrobacterium tumor-inducing
(Ti) plasmid genes, such as the nopaline synthase (NOS) gene and
(2) plant genes such as the pea ssRUBISCO E9 gene (Fischhoff et
al., 1987).
5.37.2 Plant Transformation and Expression
A chimeric plant gene containing a structural coding sequence of
the present invention can be inserted into the genome of a plant by
any suitable method. Suitable plant transformation vectors include
those derived from a Ti plasmid of Agrobacterium tumefaciens, as
well as those disclosed, e.g., by Herrera-Estrella (1983), Bevan
(1983), Klee (1985) and Eur. Pat. Appl. Publ. No. EP0120516. In
addition to plant transformation vectors derived from the Ti or
root-inducing (Ri) plasmids of Agrobacterium, alternative methods
can be used to insert the DNA constructs of this invention into
plant cells. Such methods may involve, for example, the use of
liposomes, electroporation, chemicals that increase free DNA
uptake, free DNA delivery via microprojectile bombardment, and
transformation using viruses or pollen (Fromm et al, 1986;
Armstrong et al., 1990; From et al., 1990).
5.37.3 Construction of Monocot Plant Expression Vectors for Cry3Bb
Variants
5.37.3.1 Design of Cry3Bb Variant Genes for Plant Expression
For efficient expression of the cry3Bb variants in transgenic
plants, the gene encoding the variants must have a suitable
sequence composition (Diehn et al., 1996). One example of such a
sequence is shown for the v11231 gene (SEQ ID NO:99) which encodes
the Cry3Bb11231 variant protein (SEQ ID NO:100) with Diabrotica
activity. This gene was derived via mutagenesis (Kunkel, 1985) of a
cry3Bb synthetic gene (SEQ ID NO:101) encoding a protein
essentially homologous to the protein encoded by the native cry3Bb
gene (Gen Bank Accession Number m89794, SEQ ID NO:102). The
following oligonucleotides were used in the mutagenesis of the
original cry3Bb synthetic gene (SEQ ID NO:101) to create the v11231
gene (SEQ ID NO:99): Oligo #1: (SEQ ID NO:103)
5'-TAGGCCTCCATCCATGGCAAACCCTAACAATC-3' Oligo #2: (SEQ ID NO:104)
5'-TCCCATCTTCCTACTTACGACCCTGCAGAAATACGGTCCAAC-3' Oligo #3: (SEQ ID
NO:105) 5'-GACCTCACCTACCAAACATTCGATCTTG-3' Oligo #4: (SEQ ID
NO:106) 5'-CGAGTTCTACCGTAGGCAGCTCAAG-3'
5.37.3.2 Construction of Cry3Bb Monocot Plant Expression Vector
To place the cry3Bb variant gene v11231 in a vector suitable for
expression in monocotyledonous plants (i.e. under control of the
enhanced Cauliflower Mosaic Virus 35S promoter and link to the
hsp70 intron followed by a nopaline synthase polyadenylation site
as in U.S. Pat. No. 5,424,412, specifically incorporated herein by
reference), the vector pMON19469 was digested with NcoI and EcoRI.
The larger vector band of approximately 4.6 kb was electrophoresed,
purified, and ligated with T4 DNA ligase to the NcoI-EcoRI fragment
of approximately 2 kb containing the v11231 gene (SEQ ID NO:99).
The ligation mix was transformed into E. coli, carbenicillin
resistant colonies recovered and plasmid DNA recovered by DNA
miniprep procedures. This DNA was subjected to restriction
endonuclease analysis with enzymes such as NcoI and EcoRI
(together), NotI, and PstI to identify clones containing pMON33708
(the v11231 coding sequence fused to the hsp70 intron under control
of the enhanced CaMV35S promoter).
To place the v11231 gene in a vector suitable for recovery of
stably transformed and insect resistant plants, the 3.75-kb NotI
restriction fragment from pMON33708 containing the v11231 coding
sequence fused to the hsp70 intron under control of the enhanced
CaMV35S promoter was isolated by gel electrophoresis and
purification. This fragment was ligated with pMON30460 treated with
NotI and calf intestinal alkaline phosphatase (pMON30460 contains
the neomycin phosphotransferase coding sequence under control of
the CaMV35S promoter). Kanamycin resistant colonies were obtained
by transformation of this ligation mix into E. coli and colonies
containing pMON33710 identified by restriction endonuclease
digestion of plasmid miniprep DNAs. Restriction enzymes such as
NotI, EcoRV, HindIII, NcoI, EcoRI, and BglII can be used to
identify the appropriate clones containing the NotI fragment of
pMON33708 in the NotI site of pMON30460 (i.e. pMON33710) in the
orientation such that both genes are in tandem (i.e. the 3' end of
the v11231 expression cassette is linked to the 5' end of the nptII
expression cassette). Expression of the v11231 protein by pMON33710
in corn protoplasts was confirmed by electroporation of pMON33710
DNA into protoplasts followed by protein blot and ELISA analysis.
This vector can be introduced into the genomic DNA of corn embryos
by particle gun bombardment followed by paromomycin selection to
obtain corn plants expressing the v11231 gene essentially as
described in U.S. Pat. No. 5,424,412, specifically incorporated
herein by reference.
In this example, the vector was introduced via cobombardment with a
hygromycin resistance conferring plasmid into immature embryo
scutella (IES) of maize, followed by hygromycin selection, and
regeneration. Transgenic corn lines expressing the v11231 protein
were identified by ELISA analysis. Progeny seed from these events
were subsequently tested for protection from Diabrotica
feeding.
5.37.3.3 In Planta Performance of Cry3Bb.11231
Transformed corn plants expressing Cry3Bb.11231 protein were
challenged with western corn rootworm (WCR) larvae in both a
seedling and 10 inch pot assay. The transformed genotype was A634,
where the progeny of the RO cross by A634 was evaluated.
Observations included effect on larval development (weight), root
damage rating (RDR), and protein expression. The transformation
vector containing the cry3Bb gene was pMON33710. Treatments
included the positive and negative iso-populations for each event
and an A634 check.
The seedling assay consisted of the following steps: (i) single
seeds were placed in 1 oz cups containing potting soil; (ii) at
spiking, each seedling was infested with 4 neonate larvae; and
(iii) after infestation, seedlings were incubated for 7 days at
25.degree. C., 50% RH, and 14:10 (L:D) photo period. Adequate
moisture was added to the potting soil during the incubation period
to maintain seedling vigor.
The 10 inch pot assay consisted of the following steps: (i) single
seeds were placed in 10 inch pots containing potting soil, (ii) at
14 days post planting, each pot was infested with 800 eggs which
have been pre-incubated such that hatch would occur 5-7 days post
infestation; and (iii) after infestation, plants were incubated for
4 weeks under the same environmental conditions as the seedling
assay. Pots were both sub and top irrigated daily.
For the seedling assay, on day 7 plants were given a root damage
rating, and surviving larvae were weighed. Also at this time,
Cry3Bb protein concentrations in the roots were determined by
ELISA. The scale used for the seedling assay to assess root damage
is as follows: RDR (root damage rating) 0=no visible feeding; RDR
1=very light feeding; RDR 2=light feeding; RDR 3=moderate feeding;
RDR 4=heavy feeding; and RDR 5=very heavy feeding.
Results of the seedling assay are shown in Table 26. Plants
expressing Cry3Bb protein were completely protected by WCR feeding,
where surviving larvae within this treatment had not grown. Mean
larval weights ranged from 2.03-2.73 mg for the nonexpressing
treatments, where the surviving larval average weight was 0.11 mg
on the expressing cry3Bb treatment. Root damage ratings were 3.86
and 0.33 for the nonexpressing and expressing isopopulations,
respectively. Larval survival ranged from 75-85% for the negative
and check treatments, where only 25% of the larvae survived on the
Cry3Bb treatment.
TABLE-US-00026 TABLE 26 EFFECT OF CRY3BB EXPRESSING PLANTS ON WCR
LARVAE IN A SEEDLING ASSAY Larvae Plants Mean .+-. SD Root % Wt.
Event Treatment N (ppm) RDR .+-. SD N Surv (mg) 16 Negative 7 0.0
3.86 .+-. 0.65 21 75 2.73 .+-. 1.67 16 Positive 3 29.01 0.33 .+-.
0.45 3 25 0.11 .+-. 0.07 A634 Check 4 0.0 -- 13 81 2.03 .+-.
0.83
For the 10 inch pot assay, at 4 weeks post infestation plant height
was recorded and a root damage rating (Iowa 1-6 scale; Hills and
Peters, 1971) was given.
Results of the 10 inch pot assay are shown in Table 27. Plants
expressing Cry3Bb protein had significantly less feeding damage and
were taller than the non-expressing plants. Event 16, the higher of
the two expressing events provided nearly complete control. The
negative treatments had very high root damage ratings indicating
very high insect pressure. The positive mean root damage ratings
were 3.4 and 2.2 for event 6 and 16, respectively. Mean RDR for the
negative treatment was 5.0 and 5.6.
TABLE-US-00027 TABLE 27 EFFECT OF CRY3BB EXPRESSING CORN IN
CONTROLLING WCR LARVAL FEEDING IN A 10 INCH POT ASSAY Root Plant
Height Event Treatment N (ppm) RDR .+-. SD (cm) 6 Negative 7 0.0
5.0 .+-. 1.41 49.7 .+-. 18.72 6 Positive 5 7.0 3.4 .+-. 1.14 73.9
.+-. 8.67 16 Negative 5 0.0 5.6 .+-. 0.89 61.2 .+-. 7.75 16
Positive 5 55.0 2.2 .+-. 0.84 83.8 .+-. 7.15
In summary, corn plants expressing Cry3Bb protein have a
significant biological effect on WCR larval development as seen in
the seedling assay. When challenged with very high infestation
levels, plants expressing the Cry3Bb protein were protected from
WCR larval feeding damage as illustrated in the 10 inch pot
assay.
6.0 BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS
SEQ ID NO:1 DNA sequence of cry3Bb.11221 gene. SEQ ID NO:2 Amino
acid sequence of Cry3Bb.11221 polypeptide. SEQ ID NO:3 DNA sequence
of cry3Bb.11222 gene. SEQ ID NO:4 Amino acid sequence of
Cry3Bb.11222 polypeptide. SEQ ID NO:5 DNA sequence of cry3Bb.11223
gene. SEQ ID NO:6 Amino acid sequence of Cry3Bb.11223 polypeptide.
SEQ ID NO:7 DNA sequence of cry3Bb.11224 gene. SEQ ID NO:8 Amino
acid sequence of Cry3Bb.11224 polypeptide. SEQ ID NO:9 DNA sequence
of cry3Bb.11225 gene. SEQ ID NO:10 Amino acid sequence of
Cry3Bb.11225 polypeptide. SEQ ID NO:11 DNA sequence of cry3Bb.11226
gene. SEQ ID NO:12 Amino acid sequence of Cry3Bb.11226 polypeptide.
SEQ ID NO:13 DNA sequence of cry3Bb.11227 gene. SEQ ID NO:14 Amino
acid sequence of Cry3Bb.11227 polypeptide. SEQ ID NO:15 DNA
sequence of cry3Bb.11228 gene. SEQ ID NO:16 Amino acid sequence of
Cry3Bb.11228 polypeptide. SEQ ID NO:17 DNA sequence of cry3Bb.11229
gene. SEQ ID NO:18 Amino acid sequence of Cry3Bb.11229 polypeptide.
SEQ ID NO:19 DNA sequence of cry3Bb.11230 gene. SEQ ID NO:20 Amino
acid sequence of Cry3Bb.11230 polypeptide. SEQ ID NO:21 DNA
sequence of cry3Bb.11231 gene. SEQ ID NO:22 Amino acid sequence of
Cry3Bb.11231 polypeptide. SEQ ID NO:23 DNA sequence of cry3Bb.11232
gene. SEQ ID NO:24 Amino acid sequence of Cry3Bb.11232 polypeptide.
SEQ ID NO:25 DNA sequence of cry3Bb.11233 gene. SEQ ID NO:26 Amino
acid sequence of Cry3Bb.11233 polypeptide. SEQ ID NO:27 DNA
sequence of cry3Bb.11234 gene. SEQ ID NO:28 Amino acid sequence of
Cry3Bb.11234 polypeptide. SEQ ID NO:29 DNA sequence of cry3Bb.11235
gene. SEQ ID NO:30 Amino acid sequence of Cry3Bb.11235 polypeptide.
SEQ ID NO:31 DNA sequence of cry3Bb.11236 gene. SEQ ID NO:32 Amino
acid sequence of Cry3Bb.11236 polypeptide. SEQ ID NO:33 DNA
sequence of cry3Bb.11237 gene. SEQ ID NO:34 Amino acid sequence of
Cry3Bb.11237 polypeptide. SEQ ID NO:35 DNA sequence of cry3Bb.11238
gene. SEQ ID NO:36 Amino acid sequence of Cry3Bb.11238 polypeptide.
SEQ ID NO:37 DNA sequence of cry3Bb.11239 gene. SEQ ID NO:38 Amino
acid sequence of Cry3Bb.11239 polypeptide. SEQ ID NO:39 DNA
sequence of cry3Bb.11241 gene. SEQ ID NO:40 Amino acid sequence of
Cry3Bb.11241 polypeptide. SEQ ID NO:41 DNA sequence of cry3Bb.11242
gene. SEQ ID NO:42 Amino acid sequence of Cry3Bb.11242 polypeptide.
SEQ ID NO:43 DNA sequence of cry3Bb.11032 gene. SEQ ID NO:44 Amino
acid sequence of Cry3Bb.11032 polypeptide. SEQ ID NO:45 DNA
sequence of cry3Bb.11035 gene. SEQ ID NO:46 Amino acid sequence of
Cry3Bb.11035 polypeptide. SEQ ID NO:47 DNA sequence of cry3Bb.11036
gene. SEQ ID NO:48 Amino acid sequence of Cry3Bb.11036 polypeptide.
SEQ ID NO:49 DNA sequence of cry3Bb.11046 gene. SEQ ID NO:50 Amino
acid sequence of Cry3Bb.11046 polypeptide. SEQ ID NO:51 DNA
sequence of cry3Bb.11048 gene. SEQ ID NO:52 Amino acid sequence of
Cry3Bb.11048 polypeptide. SEQ ID NO:53 DNA sequence of cry3Bb.11051
gene. SEQ ID NO:54 Amino acid sequence of Cry3Bb.11051 polypeptide.
SEQ ID NO:55 DNA sequence of cry3Bb.11057 gene. SEQ ID NO:56 Amino
acid sequence of Cry3Bb.11057 polypeptide. SEQ ID NO:57 DNA
sequence of cry3Bb.11058 gene. SEQ ID NO:58 Amino acid sequence of
Cry3Bb.11058 polypeptide. SEQ ID NO:59 DNA sequence of cry3Bb.11081
gene. SEQ ID NO:60 Amino acid sequence of Cry3Bb.11081 polypeptide.
SEQ ID NO:61 DNA sequence of cry3Bb.11082 gene. SEQ ID NO:62 Amino
acid sequence of Cry3Bb.11082 polypeptide. SEQ ID NO:63 DNA
sequence of cry3Bb.11083 gene. SEQ ID NO:64 Amino acid sequence of
Cry3Bb.11083 polypeptide. SEQ ID NO:65 DNA sequence of cry3Bb.11084
gene. SEQ ID NO:66 Amino acid sequence of Cry3Bb.11084 polypeptide.
SEQ ID NO:67 DNA sequence of cry3Bb.11095 gene. SEQ ID NO:68 Amino
acid sequence of Cry3Bb.11095 polypeptide. SEQ ID NO:69 DNA
sequence of cry3Bb.60 gene. SEQ ID NO:70 Amino acid sequence of
Cry3Bb.60 polypeptide. SEQ ID NO:71 Primer FW001. SEQ ID NO:72
Primer FW006. SEQ ID NO:73 Primer MVT095. SEQ ID NO:74 Primer
MVT097. SEQ ID NO:75 Primer MVT091. SEQ ID NO:76 Primer MVT075. SEQ
ID NO:77 Primer MVT076. SEQ ID NO:78 Primer MVT111. SEQ ID NO:79
Primer MVT094. SEQ ID NO:80 Primer MVT103. SEQ ID NO:81 Primer
MVT081. SEQ ID NO:82 Primer MVT085. SEQ ID NO:83 Primer A. SEQ ID
NO:84 Primer B. SEQ ID NO:85 Primer C. SEQ ID NO:86 Primer D. SEQ
ID NO:87 Primer E. SEQ ID NO:88 Primer F. SEQ ID NO:89 Primer G.
SEQ ID NO:90 Primer WD112. SEQ ID NO:91 Primer WD115. SEQ ID NO:92
Primer MVT105. SEQ ID NO:93 Primer MVT092. SEQ ID NO:94 Primer
MVT070. SEQ ID NO:95 Primer MVT083. SEQ ID NO:96 N-terminal amino
acid of Cry3Bb polypeptide. SEQ ID NO:97 DNA sequence of wild-type
cry3Bb gene. SEQ ID NO:98 Amino acid sequence of wild-type Cry3Bb
polypeptide. SEQ ID NO:99 Plantized DNA sequence for cry3Bb.11231
gene. SEQ ID NO:100 Amino acid sequence of plantized Cry3Bb.11231
polypeptide. SEQ ID NO:101 DNA sequence of cry3Bb gene used to
prepare SEQ ID NO:99. SEQ ID NO:102 DNA sequence of wild-type
cry3Bb gene, Genbank #M89794. SEQ ID NO:103 DNA sequence of Oligo
#1. SEQ ID NO:104 DNA sequence of Oligo #2. SEQ ID NO:105 DNA
sequence of Oligo #3. SEQ ID NO:106 DNA sequence of Oligo #4. SEQ
ID NO:107 DNA sequence of cry3Bb.11098 gene. SEQ ID NO:108 Amino
acid sequence of Cry3Bb.11098 polypeptide.
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All of the compositions and methods disclosed and claimed herein
can be made and executed without undue experimentation in light of
the present disclosure. While the compositions and methods of this
invention have been described in terms of preferred embodiments, it
will be apparent to those of skill in the art that variations may
be applied to the compositions and methods and in the steps or in
the sequence of steps of the method described herein without
departing from the concept, spirit and scope of the invention. More
specifically, it will be apparent that certain agents which are
both chemically and physiologically related may be substituted for
the agents described herein while the same or similar results would
be achieved. All such similar substitutes and modifications
apparent to those skilled in the art are deemed to be within the
spirit, scope and concept of the invention as defined by the
appended claims.
SEQUENCE LISTINGS
1
11311959DNAArtificial sequenceRecombinant delta endotoxin 1atg aat
cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met Asn
Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15aac
agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat 96Asn
Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25
30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga atg
144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met
35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta aaa
gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys
Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att tta ggt
gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly
Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca ttt tat
caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr
Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gat gct gac cca tgg
aag gct ttt atg gca 336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp
Lys Ala Phe Met Ala 100 105 110caa gtt gaa gta ctg ata gat aag aaa
ata gag gag tat gct aaa agt 384Gln Val Glu Val Leu Ile Asp Lys Lys
Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa gct ctt gca gag tta cag
ggt ctt caa aat aat ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu Gln
Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140gtt aat gcg tta aat
tcc tgg aag aaa ttt cac cat tct cgt cgt tct 480Val Asn Ala Leu Asn
Ser Trp Lys Lys Phe His His Ser Arg Arg Ser145 150 155 160aaa aga
agc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt 528Lys Arg
Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg
576His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta
ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga
tat tct tca gaa 672Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220gat gtt gct gaa ttt tat cat aga caa tta
aaa ctt aca caa caa tac 720Asp Val Ala Glu Phe Tyr His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240act gac cat tgt gtt aat tgg
tat aat gtt gga tta aat ggt tta aga 768Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act tat gat
gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg act tta
act gta tta gat cta att gta ctt ttc cca ttt tat gat 864Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285att
cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300ttt acg gat cca att ttt tca ctt aat act ctt cag gag tat gga cca
960Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly
Pro305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct
cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt
ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg
tct ggt aat tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt
aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa
cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400tat
cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa
1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac
aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445cat gta agt gca cag gat tct att gac caa tta ccg
cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat agt cat
cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag gac cgt
cgt gga aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta
gac ttt ttt aat aca att gat gct gaa aag att 1536Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt
cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att
att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca
1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560gcc ttg tta caa cga tat cgt gta aga ata cgc tat
gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca aac
aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590tac att aat aaa act atg aat aaa
gat gat gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act act
aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata
ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640tat
ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys
Ile Glu Phe Ile Pro Val Gln Leu 645 6502652PRTArtificial
sequenceRecombinant delta endotoxin 2Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Phe
His His Ser Arg Arg Ser145 150 155 160Lys Arg Ser Gln Asp Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile Arg Leu Tyr Ser Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro
Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro305 310 315 320Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe
340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His
Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455
460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630 635 640Tyr Ile Asp Lys Ile Glu Phe
Ile Pro Val Gln Leu 645 65031959DNAArtificial sequenceRecombinant
delta endotoxin 3atg aat cca aac aat cga agt gaa cat gat acg ata
aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile
Lys Val Thr Pro1 5 10 15aac agt gaa ttg caa act aac cat aat caa tat
cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr
Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat
aaa gaa ttt tta aga atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr
Lys Glu Phe Leu Arg Met 35 40 45act gaa gac agt tct acg gaa gtg cta
gac aac tct aca gta aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu
Asp Asn Ser Thr Val Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt
gta ggg cag att tta ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val
Val Gly Gln Ile Leu Gly Val Val65 70 75 80gga gtt cca ttt gct ggg
gca ctc act tca ttt tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly
Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca
agt gat gct gac cca tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro
Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa
gta ctg ata gat aag aaa ata gag gag tat gct aaa agt 384Gln Val Glu
Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa
gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat 432Lys
Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135
140gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg cga agt
480Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg
Ser145 150 155 160aaa aga agc caa gat cga ata agg gaa ctt ttt tct
caa gca gaa agt 528Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser
Gln Ala Glu Ser 165 170 175cat ttt cgt aat tcc atg ccg tca ttt gca
gtt tcc aaa ttc gaa gtg 576His Phe Arg Asn Ser Met Pro Ser Phe Ala
Val Ser Lys Phe Glu Val 180 185 190ctg ttt cta cca aca tat gca caa
gct gca aat aca cat tta ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln
Ala Ala Asn Thr His Leu Leu Leu 195 200 205tta aaa gat gct caa gtt
ttt gga gaa gaa tgg gga tat tct tca gaa 672Leu Lys Asp Ala Gln Val
Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220gat gtt gct gaa
ttc ctt agt aga caa tta aaa ctt aca caa caa tac 720Asp Val Ala Glu
Phe Leu Ser Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240act
gac cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga 768Thr
Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250
255ggt tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa
816Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu
260 265 270atg act tta act gta tta gat cta att gta ctt ttc cca ttt
tat gat 864Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe
Tyr Asp 275 280 285att cgg tta tac tca aaa ggg gtt aaa aca gaa cta
aca aga gac att 912Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu
Thr Arg Asp Ile 290 295 300ttt acg gat cca att ttt tca ctt aat act
ctt cag gag tat gga cca 960Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr
Leu Gln Glu Tyr Gly Pro305 310 315 320act ttt ttg agt ata gaa aac
tct att cga aaa cct cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn
Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335tat tta cag ggg att
gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile
Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat
tct ttc aat tat tgg tct ggt aat tat gta gaa act aga 1104Gly Lys Asp
Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct
agt ata gga tct agt aag aca att act tcc cca ttt tat gga gat 1152Pro
Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375
380aaa tct act gaa cct gta caa aag cta agc ttt gat gga caa aaa gtt
1200Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys
Val385 390 395 400tat cga act ata gct aat aca gac gta gcg gct tgg
ccg aat ggt aag 1248Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp
Pro Asn Gly Lys 405 410 415gta tat tta ggt gtt acg aaa gtt gat ttt
agt caa tat gat gat caa 1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe
Ser Gln Tyr Asp Asp Gln 420 425 430aaa aat gaa act agt aca caa aca
tat gat tca aaa aga aac aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr
Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445cat gta agt gca cag gat
tct att gac caa tta ccg cca gaa aca aca 1392His Val Ser Ala Gln Asp
Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460gat gaa cca ctt
gaa aaa gca tat agt cat cag ctt aat tac gcg gaa
1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480tgt ttc tta atg cag gac cgt cgt gga aca att cca
ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495aca cat aga agt gta gac ttt ttt aat aca
att gat gct gaa aag att 1536Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt cca gta gtg aaa gca
tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att att gaa ggt cca gga
ttc aca gga gga aat tta cta ttc cta aaa 1632Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540gaa tct agt aat
tca att gct aaa ttt aaa gtt aca tta aat tca gca 1680Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560gcc
ttg tta caa cga tat cgt gta aga ata cgc tat gct tct acc act 1728Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575aac tta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtc atc
1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590tac att aat aaa act atg aat aaa gat gat gat tta aca tat
caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605ttt gat ctc gca act act aat tct aat atg ggg ttc
tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620aat gaa ctt ata ata gga gca gaa tct ttc
gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630 635 640tat ata gat aag ata gaa ttt
atc cca gta caa ttg taa 1959Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val
Gln Leu 645 6504652PRTArtificial sequenceRecombinant delta
endotoxin 4Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val
Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu
Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu
Phe Leu Arg Met 35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn
Ser Thr Val Lys Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val Val Gly
Gln Ile Leu Gly Val Val65 70 75 80Gly Val Pro Phe Ala Gly Ala Leu
Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95Asn Thr Ile Trp Pro Ser Asp
Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu
Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125Lys Ala Leu
Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140Val
Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150
155 160Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu
Ser 165 170 175His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys
Phe Glu Val 180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn
Thr His Leu Leu Leu 195 200 205Leu Lys Asp Ala Gln Val Phe Gly Glu
Glu Trp Gly Tyr Ser Ser Glu 210 215 220Asp Val Ala Glu Phe Leu Ser
Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240Thr Asp His Cys
Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255Gly Ser
Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265
270Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp
275 280 285Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg
Asp Ile 290 295 300Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln
Glu Tyr Gly Pro305 310 315 320Thr Phe Leu Ser Ile Glu Asn Ser Ile
Arg Lys Pro His Leu Phe Asp 325 330 335Tyr Leu Gln Gly Ile Glu Phe
His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe
Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365Pro Ser Ile
Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380Lys
Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390
395 400Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly
Lys 405 410 415Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr
Asp Asp Gln 420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser
Lys Arg Asn Asn Gly 435 440 445His Val Ser Ala Gln Asp Ser Ile Asp
Gln Leu Pro Pro Glu Thr Thr 450 455 460Asp Glu Pro Leu Glu Lys Ala
Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480Cys Phe Leu Met
Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495Thr His
Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505
510Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser
515 520 525Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe
Leu Lys 530 535 540Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr
Leu Asn Ser Ala545 550 555 560Ala Leu Leu Gln Arg Tyr Arg Val Arg
Ile Arg Tyr Ala Ser Thr Thr 565 570 575Asn Leu Arg Leu Phe Val Gln
Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr
Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605Phe Asp Leu
Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620Asn
Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630
635 640Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
65051959DNAArtificial sequenceRecombinant delta endotoxin 5atg aat
cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met Asn
Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15aac
agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat 96Asn
Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25
30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga atg
144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met
35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta aaa
gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys
Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att tta ggt
gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly
Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca ttt tat
caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr
Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gat gct gac cca tgg
aag gct ttt atg gca 336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp
Lys Ala Phe Met Ala 100 105 110caa gtt gaa gta ctg ata gat aag aaa
ata gag gag tat gct aaa agt 384Gln Val Glu Val Leu Ile Asp Lys Lys
Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa gct ctt gca gag tta cag
ggt ctt caa aat aat ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu Gln
Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140gtt aat gcg tta aat
tcc tgg aag aaa aca cct tta agt ttg cga agt 480Val Asn Ala Leu Asn
Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160aaa aga
agc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt 528Lys Arg
Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg
576His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta
ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga
tat tct cca gaa 672Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Pro Glu 210 215 220gat gtt gct gaa ttc agt cat aga caa tta
aaa ctt aca caa caa tac 720Asp Val Ala Glu Phe Ser His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240act gac cat tgt gtt aat tgg
tat aat gtt gga tta aat ggt tta aga 768Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act tat gat
gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg act tta
act gta tta gat cta att gta ctt ttc cca ttt tat gat 864Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285att
cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300ttt acg gat cca att ttt tca ctt aat act ctt cag gag tat gga cca
960Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly
Pro305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct
cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt
ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg
tct ggt aat tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt
aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa
cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400tat
cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa
1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac
aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445cat gta agt gca cag gat tct att gac caa tta ccg
cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat agt cat
cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag gac cgt
cgt gga aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta
gac ttt ttt aat aca att gat gct gaa aag att 1536Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt
cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att
att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca
1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560gcc ttg tta caa cga tat cgt gta aga ata cgc tat
gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca aac
aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590tac att aat aaa act atg aat aaa
gat gat gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act act
aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata
ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640tat
ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys
Ile Glu Phe Ile Pro Val Gln Leu 645 6506652PRTArtificial
sequenceRecombinant delta endotoxin 6Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg Ser Gln Asp Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Pro Glu 210 215
220Asp Val Ala Glu Phe Ser His Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile Arg Leu Tyr Ser Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro
Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro305 310 315 320Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe
340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His
Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450
455 460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630 635 640Tyr Ile Asp Lys Ile Glu Phe
Ile Pro Val Gln Leu 645 65071959DNAArtificial sequenceRecombinant
delta endotoxin 7atg aat cca aac aat cga agt gaa cat gat acg ata
aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile
Lys Val Thr Pro1 5 10 15aac agt gaa ttg caa act aac cat aat caa tat
cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr
Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat
aaa gaa ttt tta aga atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr
Lys Glu Phe Leu Arg Met 35 40 45act gaa gac agt tct acg gaa gtg cta
gac aac tct aca gta aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu
Asp Asn Ser Thr Val Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt
gta ggg cag att tta ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val
Val Gly Gln Ile Leu Gly Val Val65 70 75 80gga gtt cca ttt gct ggg
gca ctc act tca ttt tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly
Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca
agt gat gct gac cca tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro
Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa
gta ctg ata gat aag aaa ata gag gag tat gct aaa agt 384Gln Val Glu
Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa
gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat 432Lys
Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135
140gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg cga agt
480Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg
Ser145 150 155 160aaa aga agc caa gat cga ata agg gaa ctt ttt tct
caa gca gaa agt 528Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser
Gln Ala Glu Ser 165 170 175cat ttt cgt aat tcc atg ccg tca ttt gca
gtt tcc aaa ttc gaa gtg 576His Phe Arg Asn Ser Met Pro Ser Phe Ala
Val Ser Lys Phe Glu Val 180 185 190ctg ttt cta cca aca tat gca caa
gct gca aat aca cat tta ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln
Ala Ala Asn Thr His Leu Leu Leu 195 200 205tta aaa gat gct caa gtt
ttt gga gaa gaa tgg gga tat tct tca gaa 672Leu Lys Asp Ala Gln Val
Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220gat gtt gct gaa
ttc tat cgt aga caa tta aaa ctt aca caa caa tac 720Asp Val Ala Glu
Phe Tyr Arg Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240act
gac cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga 768Thr
Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250
255ggt tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa
816Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu
260 265 270atg act tta act gta tta gat cta att gta ctt ttc cca ttt
tat gat 864Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe
Tyr Asp 275 280 285att cgg tta tac tca aaa ggg gtt aaa aca gaa cta
aca aga gac att 912Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu
Thr Arg Asp Ile 290 295 300ttt acg gat cca att ttt tca ctt aat act
ctt cag gag tat gga cca 960Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr
Leu Gln Glu Tyr Gly Pro305 310 315 320act ttt ttg agt ata gaa aac
tct att cga aaa cct cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn
Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335tat tta cag ggg att
gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile
Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat
tct ttc aat tat tgg tct ggt aat tat gta gaa act aga 1104Gly Lys Asp
Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct
agt ata gga tct agt aag aca att act tcc cca ttt tat gga gat 1152Pro
Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375
380aaa tct act gaa cct gta caa aag cta agc ttt gat gga caa aaa gtt
1200Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys
Val385 390 395 400tat cga act ata gct aat aca gac gta gcg gct tgg
ccg aat ggt aag 1248Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp
Pro Asn Gly Lys 405 410 415gta tat tta ggt gtt acg aaa gtt gat ttt
agt caa tat gat gat caa 1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe
Ser Gln Tyr Asp Asp Gln 420 425 430aaa aat gaa act agt aca caa aca
tat gat tca aaa aga aac aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr
Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445cat gta agt gca cag gat
tct att gac caa tta ccg cca gaa aca aca 1392His Val Ser Ala Gln Asp
Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460gat gaa cca ctt
gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu
Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480tgt
ttc tta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg 1488Cys
Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490
495aca cat aga agt gta gac ttt ttt aat aca att gat gct gaa aag att
1536Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile
500 505 510act caa ctt cca gta gtg aaa gca tat gcc ttg tct tca ggt
gct tcc 1584Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly
Ala Ser 515 520 525att att gaa ggt cca gga ttc aca gga gga aat tta
cta ttc cta aaa 1632Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu
Leu Phe Leu Lys 530 535 540gaa tct agt aat tca att gct aaa ttt aaa
gtt aca tta aat tca gca 1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys
Val Thr Leu Asn Ser Ala545 550 555 560gcc ttg tta caa cga tat cgt
gta aga ata cgc tat gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg
Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt
gtg caa aat tca aac aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe
Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590tac att aat
aaa act atg aat aaa gat gat gat tta aca tat caa aca 1824Tyr Ile Asn
Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt
gat ctc gca act act aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe
Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615
620aat gaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaa atc
1920Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys
Ile625 630 635 640tat ata gat aag ata gaa ttt atc cca gta caa ttg
taa 1959Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
6508652PRTArtificial sequenceRecombinant delta endotoxin 8Met Asn
Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn
Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25
30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met
35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys
Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly
Val Val65 70 75 80Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr
Gln Ser Phe Leu 85 90 95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp
Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys
Ile Glu Glu Tyr Ala Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln
Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn
Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg
Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220Asp Val Ala Glu Phe Tyr Arg Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly
Pro305 310 315 320Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640Tyr
Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
65091959DNAArtificial sequenceRecombinant delta endotoxin 9atg aat
cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met Asn
Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15aac
agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat 96Asn
Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25
30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga atg
144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met
35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta aaa
gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys
Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att tta ggt
gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly
Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca ttt tat
caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr
Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gat gct gac cca tgg
aag gct ttt atg gca 336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp
Lys Ala Phe Met Ala 100 105 110caa gtt gaa gta ctg ata gat aag aaa
ata gag gag tat gct aaa agt 384Gln Val Glu Val Leu Ile Asp Lys Lys
Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa gct ctt gca gag tta cag
ggt ctt caa aat aat ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu Gln
Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140gtt aat gcg tta aat
tcc tgg aag aaa aca cct tta agt ttg cga agt 480Val Asn Ala Leu Asn
Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160aaa aga
agc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt 528Lys Arg
Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg
576His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta
ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga
tat tct tca gaa 672Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220gat gtt gct gaa ttc tat aat aga caa tta
aaa ctt aca caa caa tac 720Asp Val Ala Glu Phe Tyr Asn Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240tct gac cat tgt gtt aat tgg
tat aat gtt gga tta aat ggt tta aga 768Ser Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act tat gat
gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg act tta
act gta tta gat cta att gta ctt ttc cca ttt tat gat 864Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285att
cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300ttt acg gat cca att ttt tca ctt aat act ctt cag gag tat gga cca
960Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly
Pro305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct
cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt
ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr
Arg
Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg
tct ggt aat tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt
aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa
cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400tat
cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa
1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac
aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445cat gta agt gca cag gat tct att gac caa tta ccg
cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat agt cat
cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag gac cgt
cgt gga aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta
gac ttt ttt aat aca att gat gct gaa aag att 1536Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt
cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att
att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca
1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560gcc ttg tta caa cga tat cgt gta aga ata cgc tat
gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca aac
aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590tac att aat aaa act atg aat aaa
gat gat gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act act
aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata
ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640tat
ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys
Ile Glu Phe Ile Pro Val Gln Leu 645 65010652PRTArtificial
sequenceRecombinant delta endotoxin 10Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg Ser Gln Asp Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220Asp Val Ala Glu Phe Tyr Asn Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240Ser Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile Arg Leu Tyr Ser Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro
Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro305 310 315 320Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe
340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His
Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455
460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630 635 640Tyr Ile Asp Lys Ile Glu Phe
Ile Pro Val Gln Leu 645 650111959DNAArtificial sequenceRecombinant
delta endotoxin 11atg aat cca aac aat cga agt gaa cat gat acg ata
aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile
Lys Val Thr Pro1 5 10 15aac agt gaa ttg caa act aac cat aat caa tat
cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr
Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat
aaa gaa ttt tta aga atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr
Lys Glu Phe Leu Arg Met 35 40 45act gaa gac agt tct acg gaa gtg cta
gac aac tct aca gta aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu
Asp Asn Ser Thr Val Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt
gta ggg cag att tta ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val
Val Gly Gln Ile Leu Gly Val Val65 70 75 80gga gtt cca ttt gct ggg
gca ctc act tca ttt tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly
Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca
agt gat gct gac cca tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro
Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa
gta ctg ata gat aag aaa ata gag gag tat gct aaa agt 384Gln Val Glu
Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa
gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat 432Lys
Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135
140gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg cga agt
480Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg
Ser145 150 155 160aaa aga agc caa gat cga ata agg gaa ctt ttt tct
caa gca gaa agt 528Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser
Gln Ala Glu Ser 165 170 175cat ttt cgt aat tcc atg ccg tca ttt gca
gtt tcc aaa ttc gaa gtg 576His Phe Arg Asn Ser Met Pro Ser Phe Ala
Val Ser Lys Phe Glu Val 180 185 190ctg ttt cta cca aca tat gca caa
gct gca aat aca cat tta ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln
Ala Ala Asn Thr His Leu Leu Leu 195 200 205tta aaa gat gct caa gtt
ttt gga gaa gaa tgg gga tat tct tca gaa 672Leu Lys Asp Ala Gln Val
Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220gat gtt gct gaa
ttc tat acc aga caa tta aaa ctt aca caa caa tac 720Asp Val Ala Glu
Phe Tyr Thr Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240act
gac cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga 768Thr
Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250
255ggt tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa
816Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu
260 265 270atg act tta act gta tta gat cta att gta ctt ttc cca ttt
tat gat 864Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe
Tyr Asp 275 280 285att cgg tta tac tca aaa ggg gtt aaa aca gaa cta
aca aga gac att 912Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu
Thr Arg Asp Ile 290 295 300ttt acg gat cca att ttt tca ctt aat act
ctt cag gag tat gga cca 960Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr
Leu Gln Glu Tyr Gly Pro305 310 315 320act ttt ttg agt ata gaa aac
tct att cga aaa cct cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn
Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335tat tta cag ggg att
gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile
Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat
tct ttc aat tat tgg tct ggt aat tat gta gaa act aga 1104Gly Lys Asp
Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct
agt ata gga tct agt aag aca att act tcc cca ttt tat gga gat 1152Pro
Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375
380aaa tct act gaa cct gta caa aag cta agc ttt gat gga caa aaa gtt
1200Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys
Val385 390 395 400tat cga act ata gct aat aca gac gta gcg gct tgg
ccg aat ggt aag 1248Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp
Pro Asn Gly Lys 405 410 415gta tat tta ggt gtt acg aaa gtt gat ttt
agt caa tat gat gat caa 1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe
Ser Gln Tyr Asp Asp Gln 420 425 430aaa aat gaa act agt aca caa aca
tat gat tca aaa aga aac aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr
Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445cat gta agt gca cag gat
tct att gac caa tta ccg cca gaa aca aca 1392His Val Ser Ala Gln Asp
Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460gat gaa cca ctt
gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu
Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480tgt
ttc tta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg 1488Cys
Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490
495aca cat aga agt gta gac ttt ttt aat aca att gat gct gaa aag att
1536Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile
500 505 510act caa ctt cca gta gtg aaa gca tat gcc ttg tct tca ggt
gct tcc 1584Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly
Ala Ser 515 520 525att att gaa ggt cca gga ttc aca gga gga aat tta
cta ttc cta aaa 1632Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu
Leu Phe Leu Lys 530 535 540gaa tct agt aat tca att gct aaa ttt aaa
gtt aca tta aat tca gca 1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys
Val Thr Leu Asn Ser Ala545 550 555 560gcc ttg tta caa cga tat cgt
gta aga ata cgc tat gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg
Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt
gtg caa aat tca aac aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe
Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590tac att aat
aaa act atg aat aaa gat gat gat tta aca tat caa aca 1824Tyr Ile Asn
Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt
gat ctc gca act act aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe
Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615
620aat gaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaa atc
1920Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys
Ile625 630 635 640tat ata gat aag ata gaa ttt atc cca gta caa ttg
taa 1959Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
65012652PRTArtificial sequenceRecombinant delta endotoxin 12Met Asn
Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn
Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25
30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met
35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys
Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly
Val Val65 70 75 80Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr
Gln Ser Phe Leu 85 90 95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp
Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys
Ile Glu Glu Tyr Ala Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln
Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn
Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg
Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220Asp Val Ala Glu Phe Tyr Thr Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr
Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly
Pro305 310 315 320Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640Tyr
Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
650131959DNAArtificial sequenceRecombinant delta endotoxin 13atg
aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met
Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10
15aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat
96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn
20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga
atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg
Met 35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta
aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val
Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att tta
ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu
Gly Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca ttt
tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe
Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gat gct gac cca
tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro
Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa gta ctg ata gat aag
aaa ata gag gag tat gct aaa agt 384Gln Val Glu Val Leu Ile Asp Lys
Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa gct ctt gca gag tta
cag ggt ctt caa aat aat ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu
Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140gtt aat gcg tta
aat tcc tgg aag aaa aca cct tta agt ttg cga agt 480Val Asn Ala Leu
Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160aaa
aga agc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt 528Lys
Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg
576His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta
ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga
tat tct tca gaa 672Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220gat gtt gct gaa ttt tat cat aga caa tta
aaa ctt aca caa caa tac 720Asp Val Ala Glu Phe Tyr His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240act gac cat tgt gtt aat tgg
tat aat gtt gga tta aat ggt tta aga 768Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act tat gat
gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg act tta
act gta tta gat cta att gta ctt ttc cca ttt tat gat 864Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285att
aat tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912Ile
Asn Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300ttt acg gat cca att ttt tca ctt aat act ctt cag gag tat gga cca
960Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly
Pro305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct
cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt
ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg
tct ggt aat tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt
aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa
cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400tat
cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa
1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac
aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445cat gta agt gca cag gat tct att gac caa tta ccg
cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat agt cat
cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag gac cgt
cgt gga aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta
gac ttt ttt aat aca att gat gct gaa aag att 1536Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt
cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att
att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca
1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560gcc ttg tta caa cga tat cgt gta aga ata cgc tat
gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca aac
aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590tac att aat aaa act atg aat aaa
gat gat gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act act
aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata
ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640tat
ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys
Ile Glu Phe Ile Pro Val Gln Leu 645 65014652PRTArtificial
sequenceRecombinant delta endotoxin 14Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg Ser Gln Asp Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile Asn Leu Tyr Ser Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro
Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro305 310 315 320Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe
340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His
Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455
460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630 635 640Tyr Ile Asp Lys Ile Glu Phe
Ile Pro Val Gln Leu 645 650151959DNAArtificial sequenceRecombinant
delta endotoxin 15atg aat cca aac aat cga agt gaa cat gat acg ata
aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile
Lys Val Thr Pro1 5 10 15aac agt gaa ttg caa act aac cat aat caa tat
cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr
Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat
aaa gaa ttt tta aga atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr
Lys Glu Phe Leu Arg Met 35 40 45act gaa gac agt tct acg gaa gtg cta
gac aac tct aca gta aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu
Asp Asn Ser Thr Val Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt
gta ggg cag att tta ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val
Val Gly Gln Ile Leu Gly Val Val65 70 75 80gga gtt cca ttt gct ggg
gca ctc act tca ttt tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly
Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca
agt gat gct gac cca tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro
Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa
gta ctg ata gat aag aaa ata gag gag tat gct aaa agt 384Gln Val Glu
Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa
gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat 432Lys
Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135
140gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg cga agt
480Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg
Ser145 150 155 160aaa aga agc caa gat cga ata agg gaa ctt ttt tct
caa gca gaa agt 528Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser
Gln Ala Glu Ser 165 170 175cat ttt cgt aat tcc atg ccg tca ttt gca
gtt tcc aaa ttc gaa gtg 576His Phe Arg Asn Ser Met Pro Ser Phe Ala
Val Ser Lys Phe Glu Val 180 185 190ctg ttt cta cca aca tat gca caa
gct gca aat aca cat tta ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln
Ala Ala Asn Thr His Leu Leu Leu 195 200 205tta aaa gat gct caa gtt
ttt gga gaa gaa tgg gga tat tct tca gaa 672Leu Lys Asp Ala Gln Val
Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220gat gtt gct gaa ttt tat cat aga caa tta aaa ctt aca caa caa tac
720Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240act gac cat tgt gtt aat tgg tat aat gtt gga tta
aat ggt tta aga 768Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255ggt tca act tat gat gca tgg gtc aaa ttt
aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270atg act tta act gta tta gat cta
att gta ctt ttc cca ttt tat gat 864Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285att cgg tta tac tca aaa
ggg gtt aaa aca gaa cta aca aga gac att 912Ile Arg Leu Tyr Ser Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300ttt acg gat cca
att ttt tta ctt act acg ctt cag aag tac gga cca 960Phe Thr Asp Pro
Ile Phe Leu Leu Thr Thr Leu Gln Lys Tyr Gly Pro305 310 315 320act
ttt ttg agt ata gaa aac tct att cga aaa cct cat tta ttt gat 1008Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335tat tta cag ggg att gaa ttt cat acg cgt ctt caa cct ggt tac ttt
1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe
340 345 350ggg aaa gat tct ttc aat tat tgg tct ggt aat tat gta gaa
act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365cct agt ata gga tct agt aag aca att act tcc cca
ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380aaa tct act gaa cct gta caa aag cta agc
ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400tat cga act ata gct aat aca
gac gta gcg gct tgg ccg aat ggt aag 1248Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415gta tat tta ggt gtt
acg aaa gtt gat ttt agt caa tat gat gat caa 1296Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430aaa aat gaa
act agt aca caa aca tat gat tca aaa aga aac aat ggc 1344Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445cat
gta agt gca cag gat tct att gac caa tta ccg cca gaa aca aca 1392His
Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455
460gat gaa cca ctt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa
1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480tgt ttc tta atg cag gac cgt cgt gga aca att cca
ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495aca cat aga agt gta gac ttt ttt aat aca
att gat gct gaa aag att 1536Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt cca gta gtg aaa gca
tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att att gaa ggt cca gga
ttc aca gga gga aat tta cta ttc cta aaa 1632Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540gaa tct agt aat
tca att gct aaa ttt aaa gtt aca tta aat tca gca 1680Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560gcc
ttg tta caa cga tat cgt gta aga ata cgc tat gct tct acc act 1728Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575aac tta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtc atc
1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590tac att aat aaa act atg aat aaa gat gat gat tta aca tat
caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605ttt gat ctc gca act act aat tct aat atg ggg ttc
tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620aat gaa ctt ata ata gga gca gaa tct ttc
gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630 635 640tat ata gat aag ata gaa ttt
atc cca gta caa ttg taa 1959Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val
Gln Leu 645 65016652PRTArtificial sequenceRecombinant delta
endotoxin 16Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val
Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu
Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu
Phe Leu Arg Met 35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn
Ser Thr Val Lys Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val Val Gly
Gln Ile Leu Gly Val Val65 70 75 80Gly Val Pro Phe Ala Gly Ala Leu
Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95Asn Thr Ile Trp Pro Ser Asp
Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu
Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125Lys Ala Leu
Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140Val
Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150
155 160Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu
Ser 165 170 175His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys
Phe Glu Val 180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn
Thr His Leu Leu Leu 195 200 205Leu Lys Asp Ala Gln Val Phe Gly Glu
Glu Trp Gly Tyr Ser Ser Glu 210 215 220Asp Val Ala Glu Phe Tyr His
Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240Thr Asp His Cys
Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255Gly Ser
Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265
270Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp
275 280 285Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg
Asp Ile 290 295 300Phe Thr Asp Pro Ile Phe Leu Leu Thr Thr Leu Gln
Lys Tyr Gly Pro305 310 315 320Thr Phe Leu Ser Ile Glu Asn Ser Ile
Arg Lys Pro His Leu Phe Asp 325 330 335Tyr Leu Gln Gly Ile Glu Phe
His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe
Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365Pro Ser Ile
Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380Lys
Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390
395 400Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly
Lys 405 410 415Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr
Asp Asp Gln 420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser
Lys Arg Asn Asn Gly 435 440 445His Val Ser Ala Gln Asp Ser Ile Asp
Gln Leu Pro Pro Glu Thr Thr 450 455 460Asp Glu Pro Leu Glu Lys Ala
Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480Cys Phe Leu Met
Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495Thr His
Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505
510Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser
515 520 525Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe
Leu Lys 530 535 540Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr
Leu Asn Ser Ala545 550 555 560Ala Leu Leu Gln Arg Tyr Arg Val Arg
Ile Arg Tyr Ala Ser Thr Thr 565 570 575Asn Leu Arg Leu Phe Val Gln
Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr
Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605Phe Asp Leu
Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620Asn
Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630
635 640Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
650171959DNAArtificial sequenceRecombinant delta endotoxin 17atg
aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met
Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10
15aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat
96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn
20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga
atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg
Met 35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta
aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val
Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att tta
ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu
Gly Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca ttt
tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe
Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gat gct gac cca
tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro
Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa gta ctg ata gat aag
aaa ata gag gag tat gct aaa agt 384Gln Val Glu Val Leu Ile Asp Lys
Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa gct ctt gca gag tta
cag ggt ctt caa aat aat ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu
Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140gtt aat gcg tta
aat tcc tgg aag aaa aca cct tta agt ttg cga agt 480Val Asn Ala Leu
Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160aaa
aga agc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt 528Lys
Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg
576His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta
ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga
tat tct tca gaa 672Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220gat gtt gct gaa ttt tat cat aga caa tta
aaa ctt aca caa caa tac 720Asp Val Ala Glu Phe Tyr His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240act gac cat tgt gtt aat tgg
tat aat gtt gga tta aat ggt tta aga 768Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act tat gat
gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg act tta
act gta tta gat cta att gta ctt ttc cca ttt tat gat 864Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285att
cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300ttt acg gat cca att ttt acc ctt aat aca cta cag aag tgc gga cca
960Phe Thr Asp Pro Ile Phe Thr Leu Asn Thr Leu Gln Lys Cys Gly
Pro305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct
cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt
ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg
tct ggt aat tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt
aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa
cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400tat
cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa
1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac
aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445cat gta agt gca cag gat tct att gac caa tta ccg
cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat agt cat
cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag gac cgt
cgt gga aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta
gac ttt ttt aat aca att gat gct gaa aag att 1536Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt
cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att
att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca
1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560gcc ttg tta caa cga tat cgt gta aga ata cgc tat
gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca aac
aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590tac att aat aaa act atg aat aaa
gat gat gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act act
aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata
ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640tat
ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys
Ile Glu Phe Ile Pro Val Gln Leu 645 65018652PRTArtificial
sequenceRecombinant delta endotoxin 18Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55
60Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65
70 75 80Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe
Leu 85 90 95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe
Met Ala 100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu
Tyr Ala Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln
Asn Asn Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys
Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg Ser Gln Asp
Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg
Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu
Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200
205Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu
210 215 220Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln
Gln Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly
Leu Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys
Phe Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp
Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile Arg Leu Tyr Ser
Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe Thr Asp
Pro Ile Phe Thr Leu Asn Thr Leu Gln Lys Cys Gly Pro305 310 315
320Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp
325 330 335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly
Tyr Phe 340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr
Val Glu Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr
Ser Pro Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln Lys
Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile Ala
Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Val Tyr Leu
Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys
Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440
445His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr
450 455 460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr
Ala Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile
Pro Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe Phe Asn
Thr Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys
Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu Gly Pro
Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540Glu Ser Ser
Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555
560Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr
565 570 575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu
Val Ile 580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu
Thr Tyr Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr Asn Ser Asn Met
Gly Phe Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile Ile Gly Ala Glu
Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640Tyr Ile Asp Lys Ile
Glu Phe Ile Pro Val Gln Leu 645 650191959DNAArtificial
sequenceRecombinant delta endotoxin 19atg aat cca aac aat cga agt
gaa cat gat acg ata aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15aac agt gaa ttg caa act
aac cat aat caa tat cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta
gaa gaa tta aat tat aaa gaa ttt tta aga atg 144Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45act gaa gac agt
tct acg gaa gtg cta gac aac tct aca gta aaa gat 192Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60gca gtt ggg
aca gga att tct gtt gta ggg cag att tta ggt gtt gta 240Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80gga
gtt cca ttt gct ggg gca ctc act tca ttt tat caa tca ttt ctt 288Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95aac act ata tgg cca agt gat gct gac cca tgg aag gct ttt atg gca
336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110caa gtt gaa gta ctg ata gat aag aaa ata gag gag tat gct
aaa agt 384Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125aaa gct ctt gca gag tta cag ggt ctt caa aat aat
ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140gtt aat gcg tta aat tcc tgg aag aaa aca
cct tta agt ttg cga agt 480Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Ser145 150 155 160aaa aga agc caa gat cga ata
agg gaa ctt ttt tct caa gca gaa agt 528Lys Arg Ser Gln Asp Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175cat ttt cgt aat tcc
atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg 576His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190ctg ttt cta
cca aca tat gca caa gct gca aat aca cat tta ttg cta 624Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205tta
aaa gat gct caa gtt ttt gga gaa gaa tgg gga tat tct tca gaa 672Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220gat gtt gct gaa ttt tat cat aga caa tta aaa ctt aca caa caa tac
720Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240act gac cat tgt gtt aat tgg tat aat gtt gga tta
aat ggt tta aga 768Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255ggt tca act tat gat gca tgg gtc aaa ttt
aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270atg act tta act gta tta gat cta
att gta ctt ttc cca ttt tat gat 864Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285att cgg tta tac tca aaa
ggg gtt aaa aca gaa cta aca aga gac att 912Ile Arg Leu Tyr Ser Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300ttt acg gat cca
att ttt gcc gtt aat act ctg tgg gaa tac gga cca 960Phe Thr Asp Pro
Ile Phe Ala Val Asn Thr Leu Trp Glu Tyr Gly Pro305 310 315 320act
ttt ttg agt ata gaa aac tct att cga aaa cct cat tta ttt gat 1008Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335tat tta cag ggg att gaa ttt cat acg cgt ctt caa cct ggt tac ttt
1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe
340 345 350ggg aaa gat tct ttc aat tat tgg tct ggt aat tat gta gaa
act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365cct agt ata gga tct agt aag aca att act tcc cca
ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380aaa tct act gaa cct gta caa aag cta agc
ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400tat cga act ata gct aat aca
gac gta gcg gct tgg ccg aat ggt aag 1248Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415gta tat tta ggt gtt
acg aaa gtt gat ttt agt caa tat gat gat caa 1296Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430aaa aat gaa
act agt aca caa aca tat gat tca aaa aga aac aat ggc 1344Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445cat
gta agt gca cag gat tct att gac caa tta ccg cca gaa aca aca 1392His
Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455
460gat gaa cca ctt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa
1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480tgt ttc tta atg cag gac cgt cgt gga aca att cca
ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495aca cat aga agt gta gac ttt ttt aat aca
att gat gct gaa aag att 1536Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt cca gta gtg aaa gca
tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att att gaa ggt cca gga
ttc aca gga gga aat tta cta ttc cta aaa 1632Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540gaa tct agt aat
tca att gct aaa ttt aaa gtt aca tta aat tca gca 1680Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560gcc
ttg tta caa cga tat cgt gta aga ata cgc tat gct tct acc act 1728Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575aac tta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtc atc
1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590tac att aat aaa act atg aat aaa gat gat gat tta aca tat
caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605ttt gat ctc gca act act aat tct aat atg ggg ttc
tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620aat gaa ctt ata ata gga gca gaa tct ttc
gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630 635 640tat ata gat aag ata gaa ttt
atc cca gta caa ttg taa 1959Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val
Gln Leu 645 65020652PRTArtificial sequenceRecombinant delta
endotoxin 20Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val
Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu
Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu
Phe Leu Arg Met 35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn
Ser Thr Val Lys Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val Val Gly
Gln Ile Leu Gly Val Val65 70 75 80Gly Val Pro Phe Ala Gly Ala Leu
Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95Asn Thr Ile Trp Pro Ser Asp
Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu
Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125Lys Ala Leu
Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140Val
Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150
155 160Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu
Ser 165 170 175His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys
Phe Glu Val 180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn
Thr His Leu Leu Leu 195 200 205Leu Lys Asp Ala Gln Val Phe Gly Glu
Glu Trp Gly Tyr Ser Ser Glu 210 215 220Asp Val Ala Glu Phe Tyr His
Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240Thr Asp His Cys
Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255Gly Ser
Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265
270Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp
275 280 285Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg
Asp Ile 290 295 300Phe Thr Asp Pro Ile Phe Ala Val Asn Thr Leu Trp
Glu Tyr Gly Pro305 310 315 320Thr Phe Leu Ser Ile Glu Asn Ser Ile
Arg Lys Pro His Leu Phe Asp 325 330 335Tyr Leu Gln Gly Ile Glu Phe
His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe
Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365Pro Ser Ile
Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380Lys
Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390
395 400Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly
Lys 405 410 415Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr
Asp Asp Gln 420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser
Lys Arg Asn Asn Gly 435 440 445His Val Ser Ala Gln Asp Ser Ile Asp
Gln Leu Pro Pro Glu Thr Thr 450 455 460Asp Glu Pro Leu Glu Lys Ala
Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480Cys Phe Leu Met
Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495Thr His
Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505
510Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser
515 520 525Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe
Leu Lys 530 535 540Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr
Leu Asn Ser Ala545 550 555 560Ala Leu Leu Gln Arg Tyr Arg Val Arg
Ile Arg Tyr Ala Ser Thr Thr 565 570 575Asn Leu Arg Leu Phe Val Gln
Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr
Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605Phe Asp Leu
Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620Asn
Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630
635 640Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
650211959DNAArtificial sequenceRecombinant delta endotoxin 21atg
aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met
Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10
15aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat
96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn
20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga
atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg
Met 35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta
aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val
Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att tta
ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu
Gly Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca ttt
tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe
Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gat gct gac
cca
tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro
Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa gta ctg ata gat aag
aaa ata gag gag tat gct aaa agt 384Gln Val Glu Val Leu Ile Asp Lys
Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa gct ctt gca gag tta
cag ggt ctt caa aat aat ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu
Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140gtt aat gcg tta
aat tcc tgg aag aaa aca cct tta agt ttg cga agt 480Val Asn Ala Leu
Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160aaa
aga agc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt 528Lys
Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg
576His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta
ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga
tat tct tca gaa 672Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220gat gtt gct gaa ttc tat cgt aga caa tta
aaa ctt aca caa caa tac 720Asp Val Ala Glu Phe Tyr Arg Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240act gac cat tgt gtt aat tgg
tat aat gtt gga tta aat ggt tta aga 768Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act tat gat
gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg act tta
act gta tta gat cta att gta ctt ttc cca ttt tat gat 864Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285att
cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300ttt acg gat cca att ttt tta ctt act acg ctt cag aag tac gga cca
960Phe Thr Asp Pro Ile Phe Leu Leu Thr Thr Leu Gln Lys Tyr Gly
Pro305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct
cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt
ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg
tct ggt aat tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt
aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa
cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400tat
cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa
1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac
aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445cat gta agt gca cag gat tct att gac caa tta ccg
cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat agt cat
cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag gac cgt
cgt gga aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta
gac ttt ttt aat aca att gat gct gaa aag att 1536Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt
cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att
att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca
1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560gcc ttg tta caa cga tat cgt gta aga ata cgc tat
gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca aac
aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590tac att aat aaa act atg aat aaa
gat gat gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act act
aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata
ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640tat
ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys
Ile Glu Phe Ile Pro Val Gln Leu 645 65022652PRTArtificial
sequenceRecombinant delta endotoxin 22Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg Ser Gln Asp Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220Asp Val Ala Glu Phe Tyr Arg Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile Arg Leu Tyr Ser Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro
Ile Phe Leu Leu Thr Thr Leu Gln Lys Tyr Gly Pro305 310 315 320Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe
340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His
Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455
460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630 635 640Tyr Ile Asp Lys Ile Glu Phe
Ile Pro Val Gln Leu 645 650231959DNAArtificial sequenceRecombinant
delta endotoxin 23atg aat cca aac aat cga agt gaa cat gat acg ata
aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile
Lys Val Thr Pro1 5 10 15aac agt gaa ttg caa act aac cat aat caa tat
cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr
Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat
aaa gaa ttt tta aga atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr
Lys Glu Phe Leu Arg Met 35 40 45act gaa gac agt tct acg gaa gtg cta
gac aac tct aca gta aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu
Asp Asn Ser Thr Val Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt
gta ggg cag att tta ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val
Val Gly Gln Ile Leu Gly Val Val65 70 75 80gga gtt cca ttt gct ggg
gca ctc act tca ttt tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly
Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca
agt gat gct gac cca tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro
Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa
gta ctg ata gat aag aaa ata gag gag tat gct aaa agt 384Gln Val Glu
Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa
gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat 432Lys
Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135
140gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg cga agt
480Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg
Ser145 150 155 160aaa aga agc caa gat cga ata agg gaa ctt ttt tct
caa gca gaa agt 528Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser
Gln Ala Glu Ser 165 170 175cat ttt cgt aat tcc atg ccg tca ttt gca
gtt tcc aaa ttc gaa gtg 576His Phe Arg Asn Ser Met Pro Ser Phe Ala
Val Ser Lys Phe Glu Val 180 185 190ctg ttt cta cca aca tat gca caa
gct gca aat aca cat tta ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln
Ala Ala Asn Thr His Leu Leu Leu 195 200 205tta aaa gat gct caa gtt
ttt gga gaa gaa tgg gga tat tct tca gaa 672Leu Lys Asp Ala Gln Val
Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220gat gtt gct gaa
ttt tat cat aga caa tta aaa ctt aca caa caa tac 720Asp Val Ala Glu
Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240act
gac cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga 768Thr
Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250
255ggt tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa
816Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu
260 265 270atg act tta act gta tta gat cta att gta ctt ttc cca ttt
tat gat 864Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe
Tyr Asp 275 280 285att cgg tta tac tca aaa ggg gtt aaa aca gaa cta
aca aga gac att 912Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu
Thr Arg Asp Ile 290 295 300ttt acg gat cca att ttt acg cca acc acc
cta cag gat tac gga cca 960Phe Thr Asp Pro Ile Phe Thr Pro Thr Thr
Leu Gln Asp Tyr Gly Pro305 310 315 320act ttt ttg agt ata gaa aac
tct att cga aaa cct cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn
Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335tat tta cag ggg att
gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile
Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat
tct ttc aat tat tgg tct ggt aat tat gta gaa act aga 1104Gly Lys Asp
Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct
agt ata gga tct agt aag aca att act tcc cca ttt tat gga gat 1152Pro
Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375
380aaa tct act gaa cct gta caa aag cta agc ttt gat gga caa aaa gtt
1200Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys
Val385 390 395 400tat cga act ata gct aat aca gac gta gcg gct tgg
ccg aat ggt aag 1248Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp
Pro Asn Gly Lys 405 410 415gta tat tta ggt gtt acg aaa gtt gat ttt
agt caa tat gat gat caa 1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe
Ser Gln Tyr Asp Asp Gln 420 425 430aaa aat gaa act agt aca caa aca
tat gat tca aaa aga aac aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr
Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445cat gta agt gca cag gat
tct att gac caa tta ccg cca gaa aca aca 1392His Val Ser Ala Gln Asp
Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460gat gaa cca ctt
gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu
Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480tgt
ttc tta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg 1488Cys
Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490
495aca cat aga agt gta gac ttt ttt aat aca att gat gct gaa aag att
1536Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile
500 505 510act caa ctt cca gta gtg aaa gca tat gcc ttg tct tca ggt
gct tcc 1584Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly
Ala Ser 515 520 525att att gaa ggt cca gga ttc aca gga gga aat tta
cta ttc cta aaa 1632Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu
Leu Phe Leu Lys 530 535 540gaa tct agt aat tca att gct aaa ttt aaa
gtt aca tta aat tca gca 1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys
Val Thr Leu Asn Ser Ala545 550 555 560gcc ttg tta caa cga tat cgt
gta aga ata cgc tat gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg
Val Arg Ile Arg Tyr Ala Ser Thr Thr 565
570 575aac tta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtc
atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val
Ile 580 585 590tac att aat aaa act atg aat aaa gat gat gat tta aca
tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr
Tyr Gln Thr 595 600 605ttt gat ctc gca act act aat tct aat atg ggg
ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly
Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata ata gga gca gaa tct
ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile Ile Gly Ala Glu Ser
Phe Val Ser Asn Glu Lys Ile625 630 635 640tat ata gat aag ata gaa
ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys Ile Glu Phe Ile Pro
Val Gln Leu 645 65024652PRTArtificial sequenceRecombinant delta
endotoxin 24Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val
Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu
Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu
Phe Leu Arg Met 35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn
Ser Thr Val Lys Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val Val Gly
Gln Ile Leu Gly Val Val65 70 75 80Gly Val Pro Phe Ala Gly Ala Leu
Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95Asn Thr Ile Trp Pro Ser Asp
Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu
Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125Lys Ala Leu
Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140Val
Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150
155 160Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu
Ser 165 170 175His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys
Phe Glu Val 180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn
Thr His Leu Leu Leu 195 200 205Leu Lys Asp Ala Gln Val Phe Gly Glu
Glu Trp Gly Tyr Ser Ser Glu 210 215 220Asp Val Ala Glu Phe Tyr His
Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240Thr Asp His Cys
Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255Gly Ser
Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265
270Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp
275 280 285Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg
Asp Ile 290 295 300Phe Thr Asp Pro Ile Phe Thr Pro Thr Thr Leu Gln
Asp Tyr Gly Pro305 310 315 320Thr Phe Leu Ser Ile Glu Asn Ser Ile
Arg Lys Pro His Leu Phe Asp 325 330 335Tyr Leu Gln Gly Ile Glu Phe
His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe
Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365Pro Ser Ile
Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380Lys
Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390
395 400Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly
Lys 405 410 415Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr
Asp Asp Gln 420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser
Lys Arg Asn Asn Gly 435 440 445His Val Ser Ala Gln Asp Ser Ile Asp
Gln Leu Pro Pro Glu Thr Thr 450 455 460Asp Glu Pro Leu Glu Lys Ala
Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480Cys Phe Leu Met
Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495Thr His
Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505
510Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser
515 520 525Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe
Leu Lys 530 535 540Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr
Leu Asn Ser Ala545 550 555 560Ala Leu Leu Gln Arg Tyr Arg Val Arg
Ile Arg Tyr Ala Ser Thr Thr 565 570 575Asn Leu Arg Leu Phe Val Gln
Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr
Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605Phe Asp Leu
Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620Asn
Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630
635 640Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
650251959DNAArtificial sequenceRecombinant delta endotoxin 25atg
aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met
Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10
15aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat
96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn
20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga
atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg
Met 35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta
aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val
Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att tta
ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu
Gly Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca ttt
tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe
Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gat gct gac cca
tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro
Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa gta ctg ata gat aag
aaa ata gag gag tat gct aaa agt 384Gln Val Glu Val Leu Ile Asp Lys
Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa gct ctt gca gag tta
cag ggt ctt caa aat aat ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu
Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140gtt aat gcg tta
aat tcc tgg aag aaa aca cct tta agt ttg cga agt 480Val Asn Ala Leu
Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160aaa
aga agc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt 528Lys
Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg
576His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta
ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga
tat tct tca gaa 672Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220gat gtt gct gaa ttt tat cat aga caa tta
aaa ctt aca caa caa tac 720Asp Val Ala Glu Phe Tyr His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240act gac cat tgt gtt aat tgg
tat aat gtt gga tta aat ggt tta aga 768Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act tat gat
gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg act tta
act gta tta gat cta att gta ctt ttc cca ttt tat gat 864Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285att
cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300ttt acg gat cca att ttt gcc ctg aat acc tta gac gag tac gga cca
960Phe Thr Asp Pro Ile Phe Ala Leu Asn Thr Leu Asp Glu Tyr Gly
Pro305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct
cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt
ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg
tct ggt aat tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt
aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa
cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400tat
cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa
1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac
aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445cat gta agt gca cag gat tct att gac caa tta ccg
cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat agt cat
cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag gac cgt
cgt gga aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta
gac ttt ttt aat aca att gat gct gaa aag att 1536Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt
cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att
att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca
1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560gcc ttg tta caa cga tat cgt gta aga ata cgc tat
gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca aac
aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590tac att aat aaa act atg aat aaa
gat gat gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act act
aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata
ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640tat
ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys
Ile Glu Phe Ile Pro Val Gln Leu 645 65026652PRTArtificial
sequenceRecombinant delta endotoxin 26Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg Ser Gln Asp Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile Arg Leu Tyr Ser Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro
Ile Phe Ala Leu Asn Thr Leu Asp Glu Tyr Gly Pro305 310 315 320Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe
340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His
Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455
460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620Asn Glu Leu
Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635
640Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
650271959DNAArtificial sequenceRecombinant delta endotoxin 27atg
aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met
Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10
15aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat
96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn
20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga
atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg
Met 35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta
aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val
Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att tta
ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu
Gly Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca ttt
tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe
Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gat gct gac cca
tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro
Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa gta ctg ata gat aag
aaa ata gag gag tat gct aaa agt 384Gln Val Glu Val Leu Ile Asp Lys
Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa gct ctt gca gag tta
cag ggt ctt caa aat aat ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu
Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140gtt aat gcg tta
aat tcc tgg aag aaa aca cct tta agt ttg cga agt 480Val Asn Ala Leu
Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160aaa
aga agc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt 528Lys
Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg
576His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta
ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga
tat tct tca gaa 672Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220gat gtt gct gaa ttt tat cat aga caa tta
aaa ctt aca caa caa tac 720Asp Val Ala Glu Phe Tyr His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240act gac cat tgt gtt aat tgg
tat aat gtt gga tta aat ggt tta aga 768Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act tat gat
gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg act tta
act gta tta gat cta att gta ctt ttc cca ttt tac gat 864Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285act
agg cga ttc aga aag ggg gtt aaa aca gaa cta aca aga gac att 912Thr
Arg Arg Phe Arg Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300ttt acg gat cca att ttt tca ctt aat act ctt cag gag tat gga cca
960Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly
Pro305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct
cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt
ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg
tct ggt aat tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt
aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa
cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400tat
cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa
1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac
aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445cat gta agt gca cag gat tct att gac caa tta ccg
cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat agt cat
cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag gac cgt
cgt gga aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta
gac ttt ttt aat aca att gat gct gaa aag att 1536Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt
cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att
att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca
1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560gcc ttg tta caa cga tat cgt gta aga ata cgc tat
gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca aac
aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590tac att aat aaa act atg aat aaa
gat gat gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act act
aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata
ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640tat
ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys
Ile Glu Phe Ile Pro Val Gln Leu 645 65028652PRTArtificial
sequenceRecombinant delta endotoxin 28Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg Ser Gln Asp Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Thr Arg Arg Phe Arg Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro
Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro305 310 315 320Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe
340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His
Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455
460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630 635 640Tyr Ile Asp Lys Ile Glu Phe
Ile Pro Val Gln Leu 645 650291959DNAArtificial sequenceRecombinant
delta endotoxin 29atg aat cca aac aat cga agt gaa cat gat acg ata
aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile
Lys Val Thr Pro1 5 10 15aac agt gaa ttg caa act aac cat aat caa tat
cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr
Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat
aaa gaa ttt tta aga atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr
Lys Glu Phe Leu Arg Met 35 40 45act gaa gac agt tct acg gaa gtg cta
gac aac tct aca gta aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu
Asp Asn Ser Thr Val Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt
gta ggg cag att tta ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val
Val Gly Gln Ile Leu Gly Val Val65 70 75 80gga gtt cca ttt gct ggg
gca ctc act tca ttt tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly
Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca
agt gat gct gac cca tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro
Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa
gta ctg ata gat aag aaa ata gag gag tat gct aaa agt 384Gln Val Glu
Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa
gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat 432Lys
Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135
140gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg cga agt
480Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg
Ser145 150 155 160aaa aga agc caa gat cga ata agg gaa ctt ttt tct
caa gca gaa agt 528Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser
Gln Ala Glu Ser 165 170 175cat ttt cgt aat tcc atg ccg tca ttt gca
gtt tcc aaa ttc gaa gtg 576His Phe Arg Asn Ser Met Pro Ser Phe Ala
Val Ser Lys Phe Glu Val 180 185 190ctg ttt cta cca aca tat gca caa
gct gca aat aca cat tta ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln
Ala Ala Asn Thr His Leu Leu Leu 195 200 205tta aaa gat gct caa gtt
ttt gga gaa gaa tgg gga tat tct tca gaa 672Leu Lys Asp Ala Gln Val
Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220gat gtt gct gaa
ttc tat cgt aga caa tta aaa ctt aca caa caa tac 720Asp Val Ala Glu
Phe Tyr Arg Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240act
gac cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga 768Thr
Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250
255ggt tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa
816Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu
260 265 270atg act tta act gta tta gat cta att gta ctt ttc cca ttt
tat gat 864Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe
Tyr Asp 275 280 285att cgg tta tac tca aaa ggg gtt aaa aca gaa cta
aca aga gac att 912Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu
Thr Arg Asp Ile 290 295 300ttt acg gat cca att ttt tta ctt aat act
ctt cag gag tat gga cca 960Phe Thr Asp Pro Ile Phe Leu Leu Asn Thr
Leu Gln Glu Tyr Gly Pro305 310 315 320act ttt ttg agt ata gaa aac
tct att cga aaa cct cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn
Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335tat tta cag ggg att
gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile
Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat
tct ttc aat tat tgg tct ggt aat tat gta gaa act aga 1104Gly Lys Asp
Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct
agt ata gga tct agt aag aca att act tcc cca ttt tat gga gat 1152Pro
Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375
380aaa tct act gaa cct gta caa aag cta agc ttt gat gga caa aaa gtt
1200Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys
Val385 390 395 400tat cga act ata gct aat aca gac gta gcg gct tgg
ccg aat ggt aag 1248Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp
Pro Asn Gly Lys 405 410 415gta tat tta ggt gtt acg aaa gtt gat ttt
agt caa tat gat gat caa 1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe
Ser Gln Tyr Asp Asp Gln 420 425 430aaa aat gaa act agt aca caa aca
tat gat tca aaa aga aac aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr
Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445cat gta agt gca cag gat
tct
att gac caa tta ccg cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser
Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa
aaa gca tat agt cat cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu
Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc
tta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg 1488Cys Phe
Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490
495aca cat aga agt gta gac ttt ttt aat aca att gat gct gaa aag att
1536Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile
500 505 510act caa ctt cca gta gtg aaa gca tat gcc ttg tct tca ggt
gct tcc 1584Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly
Ala Ser 515 520 525att att gaa ggt cca gga ttc aca gga gga aat tta
cta ttc cta aaa 1632Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu
Leu Phe Leu Lys 530 535 540gaa tct agt aat tca att gct aaa ttt aaa
gtt aca tta aat tca gca 1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys
Val Thr Leu Asn Ser Ala545 550 555 560gcc ttg tta caa cga tat cgt
gta aga ata cgc tat gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg
Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt
gtg caa aat tca aac aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe
Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590tac att aat
aaa act atg aat aaa gat gat gat tta aca tat caa aca 1824Tyr Ile Asn
Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt
gat ctc gca act act aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe
Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615
620aat gaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaa atc
1920Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys
Ile625 630 635 640tat ata gat aag ata gaa ttt atc cca gta caa ttg
taa 1959Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
65030652PRTArtificial sequenceRecombinant delta endotoxin 30Met Asn
Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn
Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25
30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met
35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys
Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly
Val Val65 70 75 80Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr
Gln Ser Phe Leu 85 90 95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp
Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys
Ile Glu Glu Tyr Ala Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln
Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn
Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg
Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220Asp Val Ala Glu Phe Tyr Arg Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300Phe Thr Asp Pro Ile Phe Leu Leu Asn Thr Leu Gln Glu Tyr Gly
Pro305 310 315 320Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640Tyr
Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
650311959DNAArtificial sequenceRecombinant delta endotoxin 31atg
aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met
Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10
15aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat
96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn
20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga
atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg
Met 35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta
aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val
Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att tta
ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu
Gly Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca ttt
tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe
Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gat gct gac cca
tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro
Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa gta ctg ata gat aag
aaa ata gag gag tat gct aaa agt 384Gln Val Glu Val Leu Ile Asp Lys
Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa gct ctt gca gag tta
cag ggt ctt caa aat aat ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu
Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140gtt aat gcg tta
aat tcc tgg aag aaa aca cct tta agt ttg cga agt 480Val Asn Ala Leu
Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160aaa
aga agc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt 528Lys
Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg
576His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta
ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga
tat tct tca gaa 672Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220gat gtt gct gaa ttt tat cat aga caa tta
aaa ctt aca caa caa tac 720Asp Val Ala Glu Phe Tyr His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240act gac cat tgt gtt aat tgg
tat aat gtt gga tta aat ggt tta aga 768Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act tat gat
gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg act tta
act gta tta gat cta att gta ctt ttc cca ttt tat gat 864Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285att
cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300ttt acg gat cca att ttt atc ctc aat acg cta cag gag tac gga cca
960Phe Thr Asp Pro Ile Phe Ile Leu Asn Thr Leu Gln Glu Tyr Gly
Pro305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct
cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt
ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg
tct ggt aat tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt
aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa
cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400tat
cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa
1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac
aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445cat gta agt gca cag gat tct att gac caa tta ccg
cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat agt cat
cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag gac cgt
cgt gga aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta
gac ttt ttt aat aca att gat gct gaa aag att 1536Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt
cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att
att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca
1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560gcc ttg tta caa cga tat cgt gta aga ata cgc tat
gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca aac
aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590tac att aat aaa act atg aat aaa
gat gat gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act act
aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata
ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640tat
ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys
Ile Glu Phe Ile Pro Val Gln Leu 645 65032652PRTArtificial
sequenceRecombinant delta endotoxin 32Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg Ser Gln Asp Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile Arg Leu Tyr Ser Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro
Ile Phe Ile Leu Asn Thr Leu Gln Glu Tyr Gly Pro305 310 315 320Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe
340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420
425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn
Gly 435 440 445His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro
Glu Thr Thr 450 455 460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln
Leu Asn Tyr Ala Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg
Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp
Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro
Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile
Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640Tyr
Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
650331959DNAArtificial sequenceRecombinant delta endotoxin 33atg
aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met
Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10
15aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat
96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn
20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga
atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg
Met 35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta
aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val
Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att tta
ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu
Gly Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca ttt
tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe
Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gat gct gac cca
tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro
Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa gta ctg ata gat aag
aaa ata gag gag tat gct aaa agt 384Gln Val Glu Val Leu Ile Asp Lys
Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa gct ctt gca gag tta
cag ggt ctt caa aat aat ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu
Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140gtt aat gcg tta
aat tcc tgg aag aaa aca cct tta agt ttg cga agt 480Val Asn Ala Leu
Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160aaa
aga agc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt 528Lys
Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg
576His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta
ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga
tat tct tca gaa 672Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220gat gtt gct gaa ttt tat cat aga caa tta
aaa ctt aca caa caa tac 720Asp Val Ala Glu Phe Tyr His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240act gac cat tgt gtt aat tgg
tat aat gtt gga tta aat ggt tta aga 768Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act tat gat
gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg act tta
act gta tta gat cta att gta ctt ttc cca ttt tat gat 864Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285att
cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300ttt acg gat cca att ttt atc cta cat acg ctg cag gag tac gga cca
960Phe Thr Asp Pro Ile Phe Ile Leu His Thr Leu Gln Glu Tyr Gly
Pro305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct
cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt
ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg
tct ggt aat tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt
aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa
cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400tat
cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa
1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac
aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445cat gta agt gca cag gat tct att gac caa tta ccg
cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat agt cat
cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag gac cgt
cgt gga aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta
gac ttt ttt aat aca att gat gct gaa aag att 1536Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt
cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att
att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca
1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560gcc ttg tta caa cga tat cgt gta aga ata cgc tat
gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca aac
aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590tac att aat aaa act atg aat aaa
gat gat gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act act
aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata
ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640tat
ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys
Ile Glu Phe Ile Pro Val Gln Leu 645 65034652PRTArtificial
sequenceRecombinant delta endotoxin 34Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg Ser Gln Asp Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile Arg Leu Tyr Ser Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro
Ile Phe Ile Leu His Thr Leu Gln Glu Tyr Gly Pro305 310 315 320Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe
340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His
Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455
460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630 635 640Tyr Ile Asp Lys Ile Glu Phe
Ile Pro Val Gln Leu 645 650351959DNAArtificial sequenceRecombinant
delta endotoxin 35atg aat cca aac aat cga agt gaa cat gat acg ata
aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile
Lys Val Thr Pro1 5 10 15aac agt gaa ttg caa act aac cat aat caa tat
cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr
Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat
aaa gaa ttt tta aga atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr
Lys Glu Phe Leu Arg Met 35 40 45act gaa gac agt tct acg gaa gtg cta
gac aac tct aca gta aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu
Asp Asn Ser Thr Val Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt
gta ggg cag att tta ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val
Val Gly Gln Ile Leu Gly Val Val65 70 75 80gga gtt cca ttt gct ggg
gca ctc act tca ttt tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly
Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca
agt gat gct gac cca tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro
Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa
gta ctg ata gat aag aaa ata gag gag tat gct aaa agt 384Gln Val Glu
Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa
gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat 432Lys
Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135
140gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg cga agt
480Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg
Ser145 150 155 160aaa aga agc caa gat cga ata agg gaa ctt ttt tct
caa gca gaa agt 528Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser
Gln Ala Glu Ser 165 170 175cat ttt cgt aat tcc atg ccg tca ttt gca
gtt tcc aaa ttc gaa gtg 576His Phe Arg Asn Ser Met Pro Ser Phe Ala
Val Ser Lys Phe Glu Val 180 185 190ctg ttt cta cca aca tat gca caa
gct gca aat aca cat tta ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln
Ala Ala Asn Thr His Leu Leu Leu 195 200 205tta aaa gat gct caa gtt
ttt gga gaa gaa tgg gga tat tct tca gaa 672Leu Lys Asp Ala Gln Val
Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220gat gtt gct gaa
ttt tat cat aga caa tta aaa ctt aca caa caa tac 720Asp Val Ala Glu
Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240act
gac cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga 768Thr
Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250
255ggt tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa
816Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu
260 265 270atg act tta act gta tta gat cta att gta ctt ttc cca ttt
tat gat 864Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe
Tyr Asp 275 280 285att cgg tta tac tca aaa ggg gtt aaa aca gaa cta
aca aga gac att 912Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu
Thr Arg Asp Ile 290 295 300ttt acg gat cca att ttt tcc ctc gtt aac
cta atg gtg tac gga cca 960Phe Thr Asp Pro Ile Phe Ser Leu Val Asn
Leu Met Val Tyr Gly Pro305 310 315 320act ttt ttg agt ata gaa aac
tct att cga aaa cct cat tta ttt gat 1008Thr Phe Leu Ser
Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335tat tta
cag ggg att gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056Tyr Leu
Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345
350ggg aaa gat tct ttc aat tat tgg tct ggt aat tat gta gaa act aga
1104Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg
355 360 365cct agt ata gga tct agt aag aca att act tcc cca ttt tat
gga gat 1152Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr
Gly Asp 370 375 380aaa tct act gaa cct gta caa aag cta agc ttt gat
gga caa aaa gtt 1200Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp
Gly Gln Lys Val385 390 395 400tat cga act ata gct aat aca gac gta
gcg gct tgg ccg aat ggt aag 1248Tyr Arg Thr Ile Ala Asn Thr Asp Val
Ala Ala Trp Pro Asn Gly Lys 405 410 415gta tat tta ggt gtt acg aaa
gtt gat ttt agt caa tat gat gat caa 1296Val Tyr Leu Gly Val Thr Lys
Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430aaa aat gaa act agt
aca caa aca tat gat tca aaa aga aac aat ggc 1344Lys Asn Glu Thr Ser
Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445cat gta agt
gca cag gat tct att gac caa tta ccg cca gaa aca aca 1392His Val Ser
Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460gat
gaa cca ctt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440Asp
Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470
475 480tgt ttc tta atg cag gac cgt cgt gga aca att cca ttt ttt act
tgg 1488Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr
Trp 485 490 495aca cat aga agt gta gac ttt ttt aat aca att gat gct
gaa aag att 1536Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala
Glu Lys Ile 500 505 510act caa ctt cca gta gtg aaa gca tat gcc ttg
tct tca ggt gct tcc 1584Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu
Ser Ser Gly Ala Ser 515 520 525att att gaa ggt cca gga ttc aca gga
gga aat tta cta ttc cta aaa 1632Ile Ile Glu Gly Pro Gly Phe Thr Gly
Gly Asn Leu Leu Phe Leu Lys 530 535 540gaa tct agt aat tca att gct
aaa ttt aaa gtt aca tta aat tca gca 1680Glu Ser Ser Asn Ser Ile Ala
Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560gcc ttg tta caa
cga tat cgt gta aga ata cgc tat gct tct acc act 1728Ala Leu Leu Gln
Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575aac tta
cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtc atc 1776Asn Leu
Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585
590tac att aat aaa act atg aat aaa gat gat gat tta aca tat caa aca
1824Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr
595 600 605ttt gat ctc gca act act aat tct aat atg ggg ttc tcg ggt
gat aag 1872Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly
Asp Lys 610 615 620aat gaa ctt ata ata gga gca gaa tct ttc gtt tct
aat gaa aaa atc 1920Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser
Asn Glu Lys Ile625 630 635 640tat ata gat aag ata gaa ttt atc cca
gta caa ttg taa 1959Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu
645 65036652PRTArtificial sequenceRecombinant delta endotoxin 36Met
Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10
15Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn
20 25 30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg
Met 35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val
Lys Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu
Gly Val Val65 70 75 80Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe
Tyr Gln Ser Phe Leu 85 90 95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro
Trp Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu Ile Asp Lys
Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu
Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu
Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160Lys
Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220Asp Val Ala Glu Phe Tyr His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300Phe Thr Asp Pro Ile Phe Ser Leu Val Asn Leu Met Val Tyr Gly
Pro305 310 315 320Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640Tyr
Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
650371959DNAArtificial sequenceRecombinant delta endotoxin 37atg
aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met
Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10
15aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat
96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn
20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga
atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg
Met 35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta
aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val
Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att tta
ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu
Gly Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca ttt
tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe
Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gat gct gac cca
tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro
Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa gta ctg ata gat aag
aaa ata gag gag tat gct aaa agt 384Gln Val Glu Val Leu Ile Asp Lys
Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa gct ctt gca gag tta
cag ggt ctt caa aat aat ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu
Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140gtt aat gcg tta
aat tcc tgg aag aaa aca cct tta agt ttg cga agt 480Val Asn Ala Leu
Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160aaa
aga agc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt 528Lys
Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg
576His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta
ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga
tat tct tca gaa 672Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220gat gtt gct gaa ttt tat cat aga caa tta
aaa ctt aca caa caa tac 720Asp Val Ala Glu Phe Tyr His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240act gac cat tgt gtt aat tgg
tat aat gtt gga tta aat ggt tta aga 768Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act tat gat
gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg act tta
act gta tta gat cta att gta ctt ttc cca ttt tat gat 864Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285att
cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300ttt acg gat cca att ttt tct ctt agg aca cca ctt gcg tac gga cca
960Phe Thr Asp Pro Ile Phe Ser Leu Arg Thr Pro Leu Ala Tyr Gly
Pro305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct
cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt
ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg
tct ggt aat tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt
aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa
cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400tat
cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa
1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac
aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445cat gta agt gca cag gat tct att gac caa tta ccg
cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat agt cat
cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag gac cgt
cgt gga aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta
gac ttt ttt aat aca att gat gct gaa aag att 1536Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt
cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att
att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca
1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560gcc ttg tta caa cga tat cgt gta aga ata cgc tat
gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca aac
aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590tac att aat aaa act atg aat aaa
gat gat gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act act
aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata
ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640tat
ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys
Ile Glu Phe Ile Pro Val Gln Leu 645 65038652PRTArtificial
sequenceRecombinant delta endotoxin 38Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg Ser Gln Asp Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu
Thr Gln Gln Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn
Val Gly Leu Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp
Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val
Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile Arg Leu
Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe
Thr Asp Pro Ile Phe Ser Leu Arg Thr Pro Leu Ala Tyr Gly Pro305 310
315 320Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe
Asp 325 330 335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro
Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn
Tyr Val Glu Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile
Thr Ser Pro Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln
Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile
Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Val Tyr
Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425
430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly
435 440 445His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu
Thr Thr 450 455 460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu
Asn Tyr Ala Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly
Thr Ile Pro Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe
Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val
Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu
Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540Glu
Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550
555 560Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr
Thr 565 570 575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe
Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp
Leu Thr Tyr Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr Asn Ser Asn
Met Gly Phe Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile Ile Gly Ala
Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640Tyr Ile Asp Lys
Ile Glu Phe Ile Pro Val Gln Leu 645 650391959DNAArtificial
sequenceRecombinant delta endotoxin 39atg aat cca aac aat cga agt
gaa cat gat acg ata aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15aac agt gaa ttg caa act
aac cat aat caa tat cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta
gaa gaa tta aat tat aaa gaa ttt tta aga atg 144Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45act gaa gac agt
tct acg gaa gtg cta gac aac tct aca gta aaa gat 192Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60gca gtt ggg
aca gga att tct gtt gta ggg cag att tta ggt gtt gta 240Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80gga
gtt cca ttt gct ggg gca ctc act tca ttt tat caa tca ttt ctt 288Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95aac act ata tgg cca agt gat gct gac cca tgg aag gct ttt atg gca
336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110caa gtt gaa gta ctg ata gat aag aaa ata gag gag tat gct
aaa agt 384Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125aaa gct ctt gca gag tta cag ggt ctt caa aat aat
ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140gtt aat gcg tta aat tcc tgg aag aaa aca
cct tta agt ttg cga agt 480Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Ser145 150 155 160aaa aga agc caa gat cga ata
agg gaa ctt ttt tct caa gca gaa agt 528Lys Arg Ser Gln Asp Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175cat ttt cgt aat tcc
atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg 576His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190ctg ttt cta
cca aca tat gca caa gct gca aat aca cat tta ttg cta 624Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205tta
aaa gat gct caa gtt ttt gga gaa gaa tgg gga tat tct tca gaa 672Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220gat gtt gct gaa ttt tat cat aga caa tta aaa ctt aca caa caa tac
720Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240act gac cat tgt gtt aat tgg tat aat gtt gga tta
aat ggt tta aga 768Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255ggt tca act tat gat gca tgg gtc aaa ttt
aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270atg act tta act gta tta gat cta
att gta ctt ttc cca ttt ttc aat 864Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Phe Asn 275 280 285att ttg ctt tac agt aaa
ggg gtt aaa aca gaa cta aca aga gac att 912Ile Leu Leu Tyr Ser Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300ttt acg gat cca
att ttt tca ctt aat act ctt cag gag tat gga cca 960Phe Thr Asp Pro
Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro305 310 315 320act
ttt ttg agt ata gaa aac tct att cga aaa cct cat tta ttt gat 1008Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335tat tta cag ggg att gaa ttt cat acg cgt ctt caa cct ggt tac ttt
1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe
340 345 350ggg aaa gat tct ttc aat tat tgg tct ggt aat tat gta gaa
act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365cct agt ata gga tct agt aag aca att act tcc cca
ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380aaa tct act gaa cct gta caa aag cta agc
ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400tat cga act ata gct aat aca
gac gta gcg gct tgg ccg aat ggt aag 1248Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415gta tat tta ggt gtt
acg aaa gtt gat ttt agt caa tat gat gat caa 1296Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430aaa aat gaa
act agt aca caa aca tat gat tca aaa aga aac aat ggc 1344Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445cat
gta agt gca cag gat tct att gac caa tta ccg cca gaa aca aca 1392His
Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455
460gat gaa cca ctt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa
1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480tgt ttc tta atg cag gac cgt cgt gga aca att cca
ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495aca cat aga agt gta gac ttt ttt aat aca
att gat gct gaa aag att 1536Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt cca gta gtg aaa gca
tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att att gaa ggt cca gga
ttc aca gga gga aat tta cta ttc cta aaa 1632Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540gaa tct agt aat
tca att gct aaa ttt aaa gtt aca tta aat tca gca 1680Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560gcc
ttg tta caa cga tat cgt gta aga ata cgc tat gct tct acc act 1728Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575aac tta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtc atc
1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590tac att aat aaa act atg aat aaa gat gat gat tta aca tat
caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605ttt gat ctc gca act act aat tct aat atg ggg ttc
tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620aat gaa ctt ata ata gga gca gaa tct ttc
gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630 635 640tat ata gat aag ata gaa ttt
atc cca gta caa ttg taa 1959Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val
Gln Leu 645 65040652PRTArtificial sequenceRecombinant delta
endotoxin 40Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val
Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu
Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu
Phe Leu Arg Met 35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn
Ser Thr Val Lys Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val Val Gly
Gln Ile Leu Gly Val Val65 70 75 80Gly Val Pro Phe Ala Gly Ala Leu
Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95Asn Thr Ile Trp Pro Ser Asp
Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu
Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125Lys Ala Leu
Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140Val
Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150
155 160Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu
Ser 165 170 175His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys
Phe Glu Val 180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn
Thr His Leu Leu Leu 195 200 205Leu Lys Asp Ala Gln Val Phe Gly Glu
Glu Trp Gly Tyr Ser Ser Glu 210 215 220Asp Val Ala Glu Phe Tyr His
Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240Thr Asp His Cys
Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255Gly Ser
Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265
270Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Phe Asn
275 280 285Ile Leu Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg
Asp Ile 290 295 300Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln
Glu Tyr Gly Pro305 310 315 320Thr Phe Leu Ser Ile Glu Asn Ser Ile
Arg Lys Pro His Leu Phe Asp 325 330 335Tyr Leu Gln Gly Ile Glu Phe
His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe
Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365Pro Ser Ile
Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380Lys
Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390
395 400Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly
Lys 405 410 415Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr
Asp Asp Gln 420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser
Lys Arg Asn Asn Gly 435 440 445His Val Ser Ala Gln Asp Ser Ile Asp
Gln Leu Pro Pro Glu Thr Thr 450 455 460Asp Glu Pro Leu Glu Lys Ala
Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480Cys Phe Leu Met
Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495Thr His
Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505
510Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser
515 520 525Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe
Leu Lys 530 535 540Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr
Leu Asn Ser Ala545 550 555 560Ala Leu Leu Gln Arg Tyr Arg Val Arg
Ile Arg Tyr Ala Ser Thr Thr 565 570 575Asn Leu Arg Leu Phe Val Gln
Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr
Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605Phe Asp Leu
Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620Asn
Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630
635 640Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
650411959DNAArtificial sequenceRecombinant delta endotoxin 41atg
aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met
Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10
15aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat
96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn
20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga
atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg
Met 35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta
aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val
Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att tta
ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu
Gly Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca ttt
tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe
Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gat gct gac cca
tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro
Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa gta ctg ata gat aag
aaa ata gag gag tat gct aaa agt 384Gln Val Glu Val Leu Ile Asp Lys
Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa gct ctt gca gag tta
cag ggt ctt caa aat aat ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu
Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140gtt aat gcg tta
aat tcc tgg aag aaa aca cct tta agt ttg cga agt 480Val Asn Ala Leu
Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160aaa
aga agc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt 528Lys
Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg
576His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta
ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu
195 200 205tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga tat tct
tca gaa 672Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser
Ser Glu 210 215 220gat gtt gct gaa ttt tat cat aga caa tta aaa ctt
aca caa caa tac 720Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu
Thr Gln Gln Tyr225 230 235 240act gac cat tgt gtt aat tgg tat aat
gtt gga tta aat ggt tta aga 768Thr Asp His Cys Val Asn Trp Tyr Asn
Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act tat gat gca tgg
gtc aaa ttt aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp Ala Trp
Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg act tta act gta
tta gat cta att gta ctt ttc cca ttt tat gat 864Met Thr Leu Thr Val
Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285att gtg tta
tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912Ile Val Leu
Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300ttt
acg gat cca att ttt tca ctt aat act ctt cag gag tat gga cca 960Phe
Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro305 310
315 320act ttt ttg agt ata gaa aac tct att cga aaa cct cat tta ttt
gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe
Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt ctt caa cct
ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro
Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg tct ggt aat
tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn
Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt aag aca att
act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser Lys Thr Ile
Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa cct gta caa
aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu Pro Val Gln
Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400tat cga act ata
gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr Arg Thr Ile
Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415gta tat
tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa 1296Val Tyr
Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425
430aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac aat ggc
1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly
435 440 445cat gta agt gca cag gat tct att gac caa tta ccg cca gaa
aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu
Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat agt cat cag ctt
aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu
Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag gac cgt cgt gga
aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg Arg Gly
Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta gac ttt
ttt aat aca att gat gct gaa aag att 1536Thr His Arg Ser Val Asp Phe
Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt cca gta
gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu Pro Val
Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att att gaa
ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632Ile Ile Glu
Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540gaa
tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca 1680Glu
Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550
555 560gcc ttg tta caa cga tat cgt gta aga ata cgc tat gct tct acc
act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr
Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca aac aat gat ttt
ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe
Leu Val Ile 580 585 590tac att aat aaa act atg aat aaa gat gat gat
tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp
Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act act aat tct aat
atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr Asn Ser Asn
Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata ata gga gca
gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile Ile Gly Ala
Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640tat ata gat aag
ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys Ile Glu Phe
Ile Pro Val Gln Leu 645 65042652PRTArtificial sequenceRecombinant
delta endotoxin 42Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile
Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr
Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr
Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu
Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val
Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly Val Pro Phe Ala Gly
Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95Asn Thr Ile Trp Pro
Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110Gln Val Glu
Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125Lys
Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135
140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg
Ser145 150 155 160Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser
Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser Met Pro Ser Phe Ala
Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln
Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu Lys Asp Ala Gln Val
Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220Asp Val Ala Glu
Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240Thr
Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250
255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu
260 265 270Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe
Tyr Asp 275 280 285Ile Val Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu
Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr
Leu Gln Glu Tyr Gly Pro305 310 315 320Thr Phe Leu Ser Ile Glu Asn
Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335Tyr Leu Gln Gly Ile
Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp
Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365Pro
Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375
380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys
Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp
Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val Thr Lys Val Asp Phe
Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu Thr Ser Thr Gln Thr
Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His Val Ser Ala Gln Asp
Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460Asp Glu Pro Leu
Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480Cys
Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490
495Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile
500 505 510Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly
Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu
Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys
Val Thr Leu Asn Ser Ala545 550 555 560Ala Leu Leu Gln Arg Tyr Arg
Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575Asn Leu Arg Leu Phe
Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn
Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605Phe
Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615
620Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys
Ile625 630 635 640Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu
645 650431959DNAArtificial sequenceRecombinant delta endotoxin
43atg aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct
48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1
5 10 15aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac
aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp
Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta
aga atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu
Arg Met 35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca
gta aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr
Val Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att
tta ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile
Leu Gly Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca
ttt tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser
Phe Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gat gct gac
cca tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro Ser Asp Ala Asp
Pro Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa gta ctg ata gat
aag aaa ata gag gag tat gct aaa agt 384Gln Val Glu Val Leu Ile Asp
Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa gct ctt gca gag
tta cag ggt ctt caa aat aat ttc gaa gat tat 432Lys Ala Leu Ala Glu
Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140gtt aat gcg
tta aat tcc tgg aag aaa aca cct tta agt ttg cga agt 480Val Asn Ala
Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155
160aaa aga agc caa ggt cga ata agg gaa ctt ttt tct caa gca gaa agt
528Lys Arg Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser
165 170 175cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc
gaa gtg 576His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe
Glu Val 180 185 190ctg ttt cta cca aca tat gca caa gct gca aat aca
cat tta ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr
His Leu Leu Leu 195 200 205tta aaa gat gct caa gtt ttt gga gaa gaa
tgg gga tat tct tca gaa 672Leu Lys Asp Ala Gln Val Phe Gly Glu Glu
Trp Gly Tyr Ser Ser Glu 210 215 220gat gtt gct gaa ttt tat cat aga
caa tta aaa ctt aca caa caa tac 720Asp Val Ala Glu Phe Tyr His Arg
Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240act gac cat tgt gtt
aat tgg tat aat gtt gga tta aat ggt tta aga 768Thr Asp His Cys Val
Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act
tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816Gly Ser Thr
Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg
act tta act gta tta gat cta att gta ctt ttc cca ttt tat gat 864Met
Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280
285att cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att
912Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile
290 295 300ttt acg gat cca att ttt tca ctt aat act ctt cag gag tat
gga cca 960Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr
Gly Pro305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa
cct cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys
Pro His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg
cgt ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr
Arg Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat
tgg tct ggt aat tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr
Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct
agt aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser
Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act
gaa cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr
Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395
400tat cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag
1248Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys
405 410 415gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat
gat caa 1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp
Asp Gln 420 425 430aaa aat gaa act agt aca caa aca tat gat tca aaa
aga aac aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys
Arg Asn Asn Gly 435 440 445cat gta agt gca cag gat tct att gac caa
tta ccg cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln
Leu Pro Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat
agt cat cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr
Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag
gac cgt cgt gga aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln
Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga
agt gta gac ttt ttt aat aca att gat gct gaa aag att 1536Thr His Arg
Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act
caa ctt cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr
Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520
525att att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa
1632Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys
530 535 540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat
tca gca 1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn
Ser Ala545 550 555 560gcc ttg tta caa cga tat cgt gta aga ata cgc
tat gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg
Tyr Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca
aac aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser
Asn Asn Asp Phe Leu Val Ile 580 585 590tac att aat aaa act atg aat
aaa gat gat gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn
Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act
act aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr
Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt
ata ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu
Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635
640tat ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile
Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 65044652PRTArtificial
sequenceRecombinant delta endotoxin 44Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25
30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met
35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys
Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly
Val Val65 70 75 80Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr
Gln Ser Phe Leu 85 90 95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp
Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys
Ile Glu Glu Tyr Ala Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln
Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn
Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg
Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220Asp Val Ala Glu Phe Tyr His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly
Pro305 310 315 320Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640Tyr
Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
650451959DNAArtificial sequenceRecombinant delta endotoxin 45atg
aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met
Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10
15aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat
96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn
20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga
atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg
Met 35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta
aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val
Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att tta
ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu
Gly Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca ttt
tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe
Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gat gct gac cca
tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro
Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa gta ctg ata gat aag
aaa ata gag gag tat gct aaa agt 384Gln Val Glu Val Leu Ile Asp Lys
Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa gct ctt gca gag tta
cag ggt ctt caa aat aat ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu
Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140gtt aat gcg tta
aat tcc tgg aag aaa aca cct tta agt ttg cga aat 480Val Asn Ala Leu
Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Asn145 150 155 160cca
cac agc caa ggt cga ata agg gaa ctt ttt tct caa gca gaa agt 528Pro
His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg
576His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta
ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga
tat tct tca gaa 672Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220gat gtt gct gaa ttt tat cat aga caa tta
aaa ctt aca caa caa tac 720Asp Val Ala Glu Phe Tyr His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240act gac cat tgt gtt aat tgg
tat aat gtt gga tta aat ggt tta aga 768Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act tat gat
gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg act tta
act gta tta gat cta att gta ctt ttc cca ttt tat gat 864Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285att
cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300ttt acg gat cca att ttt tca ctt aat act ctt cag gag tat gga cca
960Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly
Pro305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct
cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt
ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg
tct ggt aat tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt
aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa
cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400tat
cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa
1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac
aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445cat gta agt gca cag gat tct att gac caa tta ccg
cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat agt cat
cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag gac cgt
cgt gga aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta
gac ttt ttt aat aca att gat gct gaa aag att 1536Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt
cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att
att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca
1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560gcc ttg tta caa cga tat cgt gta aga ata cgc tat
gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca aac
aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590tac att aat aaa act atg aat aaa
gat gat gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act act
aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata
ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640tat
ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys
Ile Glu Phe Ile Pro Val Gln Leu 645 65046652PRTArtificial
sequenceRecombinant delta endotoxin 46Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Asn145 150 155 160Pro His Ser Gln Gly Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile Arg Leu Tyr Ser Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro
Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro305 310 315 320Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe
340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His
Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455
460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630 635 640Tyr Ile Asp Lys Ile Glu Phe
Ile Pro Val Gln Leu 645 650471959DNAArtificial sequenceRecombinant
delta endotoxin 47atg aat cca aac aat cga agt gaa cat gat acg ata
aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile
Lys Val Thr Pro1 5 10 15aac agt gaa ttg caa act aac cat aat caa tat
cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr
Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat
aaa gaa ttt tta aga atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr
Lys Glu Phe Leu Arg Met 35 40 45act gaa gac agt tct acg gaa gtg cta
gac aac tct aca gta aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu
Asp Asn Ser Thr Val Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt
gta ggg cag att tta ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val
Val Gly Gln Ile Leu Gly Val Val65 70 75 80gga gtt cca ttt
gct ggg gca ctc act tca ttt tat caa tca ttt ctt 288Gly Val Pro Phe
Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95aac act ata
tgg cca agt gat gct gac cca tgg aag gct ttt atg gca 336Asn Thr Ile
Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110caa
gtt gaa gta ctg ata gat aag aaa ata gag gag tat gct aaa agt 384Gln
Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120
125aaa gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat
432Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr
130 135 140gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg
cga agt 480Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu
Arg Ser145 150 155 160aaa aga agc caa gat cga ata agg gaa ctt ttt
tct caa gca gaa agt 528Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe
Ser Gln Ala Glu Ser 165 170 175cat ttt cgt aat tcc atg ccg tca ttt
gca gtt tcc aaa ttc gaa gtg 576His Phe Arg Asn Ser Met Pro Ser Phe
Ala Val Ser Lys Phe Glu Val 180 185 190ctg ttt cta cca aca tat gca
caa gct gca aat aca cat tta ttg cta 624Leu Phe Leu Pro Thr Tyr Ala
Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205tta aaa gat gct caa
gtt ttt gga gaa gaa tgg gga tat tct tca gaa 672Leu Lys Asp Ala Gln
Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220gat gtt gct
gaa ttt tat cat aga caa tta aaa ctt aca caa caa tac 720Asp Val Ala
Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235
240act gac cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga
768Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg
245 250 255ggt tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgc
aga gaa 816Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg
Arg Glu 260 265 270atg act tta act gta tta gat cta att gta ctt ttc
cca ttt tat gat 864Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe
Pro Phe Tyr Asp 275 280 285gtt cgg tta tac cca aaa ggg gtt aaa aca
gaa cta aca aga gac att 912Val Arg Leu Tyr Pro Lys Gly Val Lys Thr
Glu Leu Thr Arg Asp Ile 290 295 300ttt acg gat cca att ttt tca ctt
aat act ctt cag gag tat gga cca 960Phe Thr Asp Pro Ile Phe Ser Leu
Asn Thr Leu Gln Glu Tyr Gly Pro305 310 315 320act ttt ttg agt ata
gaa aac tct att cga aaa cct cat tta ttt gat 1008Thr Phe Leu Ser Ile
Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335tat tta cag
ggg att gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056Tyr Leu Gln
Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350ggg
aaa gat tct ttc aat tat tgg tct ggt aat tat gta gaa act aga 1104Gly
Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360
365cct agt ata gga tct agt aag aca att act tcc cca ttt tat gga gat
1152Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp
370 375 380aaa tct act gaa cct gta caa aag cta agc ttt gat gga caa
aaa gtt 1200Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln
Lys Val385 390 395 400tat cga act ata gct aat aca gac gta gcg gct
tgg ccg aat ggt aag 1248Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala
Trp Pro Asn Gly Lys 405 410 415gta tat tta ggt gtt acg aaa gtt gat
ttt agt caa tat gat gat caa 1296Val Tyr Leu Gly Val Thr Lys Val Asp
Phe Ser Gln Tyr Asp Asp Gln 420 425 430aaa aat gaa act agt aca caa
aca tat gat tca aaa aga aac aat ggc 1344Lys Asn Glu Thr Ser Thr Gln
Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445cat gta agt gca cag
gat tct att gac caa tta ccg cca gaa aca aca 1392His Val Ser Ala Gln
Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460gat gaa cca
ctt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440Asp Glu Pro
Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475
480tgt ttc tta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg
1488Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp
485 490 495aca cat aga agt gta gac ttt ttt aat aca att gat gct gaa
aag att 1536Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu
Lys Ile 500 505 510act caa ctt cca gta gtg aaa gca tat gcc ttg tct
tca ggt gct tcc 1584Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser
Ser Gly Ala Ser 515 520 525att att gaa ggt cca gga ttc aca gga gga
aat tta cta ttc cta aaa 1632Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly
Asn Leu Leu Phe Leu Lys 530 535 540gaa tct agt aat tca att gct aaa
ttt aaa gtt aca tta aat tca gca 1680Glu Ser Ser Asn Ser Ile Ala Lys
Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560gcc ttg tta caa cga
tat cgt gta aga ata cgc tat gct tct acc act 1728Ala Leu Leu Gln Arg
Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575aac tta cga
ctt ttt gtg caa aat tca aac aat gat ttt ctt gtc atc 1776Asn Leu Arg
Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590tac
att aat aaa act atg aat aaa gat gat gat tta aca tat caa aca 1824Tyr
Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600
605ttt gat ctc gca act act aat tct aat atg ggg ttc tcg ggt gat aag
1872Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys
610 615 620aat gaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa
aaa atc 1920Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu
Lys Ile625 630 635 640tat ata gat aag ata gaa ttt atc cca gta caa
ttg taa 1959Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
65048652PRTArtificial sequenceRecombinant delta endotoxin 48Met Asn
Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn
Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25
30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met
35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys
Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly
Val Val65 70 75 80Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr
Gln Ser Phe Leu 85 90 95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp
Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys
Ile Glu Glu Tyr Ala Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln
Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn
Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg
Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220Asp Val Ala Glu Phe Tyr His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Val
Arg Leu Tyr Pro Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly
Pro305 310 315 320Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640Tyr
Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
650491959DNAArtificial sequenceRecombinant delta endotoxin 49atg
aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met
Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10
15aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat
96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn
20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga
atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg
Met 35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta
aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val
Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att tta
ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu
Gly Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca ttt
tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe
Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gat gct gac cca
tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro
Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa gta ctg ata gat aag
aaa ata gag gag tat gct aaa agt 384Gln Val Glu Val Leu Ile Asp Lys
Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa gct ctt gca gag tta
cag ggt ctt caa aat aat ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu
Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140gtt aat gcg tta
aat tcc tgg aag aaa aca cct tta agt ttg cga aat 480Val Asn Ala Leu
Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Asn145 150 155 160cca
cac agc caa ggt cga ata agg gaa ctt ttt tct caa gca gaa agt 528Pro
His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg
576His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta
ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga
tat tct tca gaa 672Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220gat gtt gct gaa ttt tat cat aga caa tta
aaa ctt aca caa caa tac 720Asp Val Ala Glu Phe Tyr His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240act gac cat tgt gtt aat tgg
tat aat gtt gga tta aat ggt tta aga 768Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act tat gat
gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg act tta
act gta tta gat cta att gta ctt ttc cca ttt tat gat 864Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285gtt
cgg tta tac cca aaa ggg gtt aaa aca gaa cta aca aga gac att 912Val
Arg Leu Tyr Pro Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300ttt acg gat cca att ttt tca ctt aat act ctt cag gag tat gga cca
960Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly
Pro305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct
cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt
ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg
tct ggt aat tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt
aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa
cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400tat
cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa
1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac
aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445cat gta agt gca cag gat tct att gac caa tta ccg
cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat agt cat
cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag gac cgt
cgt gga aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta
gac ttt ttt aat aca att gat gct gaa aag att 1536Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt
cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att
att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca
1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545
550 555 560gcc ttg tta caa cga tat cgt gta aga ata cgc tat gct tct
acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser
Thr Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca aac aat gat
ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp
Phe Leu Val Ile 580 585 590tac att aat aaa act atg aat aaa gat gat
gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp
Asp Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act act aat tct
aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr Asn Ser
Asn Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata ata gga
gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile Ile Gly
Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640tat ata gat
aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys Ile Glu
Phe Ile Pro Val Gln Leu 645 65050652PRTArtificial
sequenceRecombinant delta endotoxin 50Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Asn145 150 155 160Pro His Ser Gln Gly Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Val Arg Leu Tyr Pro Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro
Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro305 310 315 320Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe
340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His
Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455
460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630 635 640Tyr Ile Asp Lys Ile Glu Phe
Ile Pro Val Gln Leu 645 650511956DNAArtificial sequenceRecombinant
delta endotoxin 51atg aat cca aac aat cga agt gaa cat gat acg ata
aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile
Lys Val Thr Pro1 5 10 15aac agt gaa ttg caa act aac cat aat caa tat
cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr
Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat
aaa gaa ttt tta aga atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr
Lys Glu Phe Leu Arg Met 35 40 45act gaa gac agt tct acg gaa gtg cta
gac aac tct aca gta aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu
Asp Asn Ser Thr Val Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt
gta ggg cag att tta ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val
Val Gly Gln Ile Leu Gly Val Val65 70 75 80gga gtt cca ttt gct ggg
gca ctc act tca ttt tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly
Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca
agt gaa gac cca tgg aag gct ttt atg gca caa 336Asn Thr Ile Trp Pro
Ser Glu Asp Pro Trp Lys Ala Phe Met Ala Gln 100 105 110gtt gaa gta
ctg ata gat aag aaa ata gag gag tat gct aaa agt aaa 384Val Glu Val
Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser Lys 115 120 125gct
ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat gtt 432Ala
Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr Val 130 135
140aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg cga agt aaa
480Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser
Lys145 150 155 160aga agc caa gat cga ata agg gaa ctt ttt tct caa
gca gaa agt cat 528Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln
Ala Glu Ser His 165 170 175ttt cgt aat tcc atg ccg tca ttt gca gtt
tcc aaa ttc gaa gtg ctg 576Phe Arg Asn Ser Met Pro Ser Phe Ala Val
Ser Lys Phe Glu Val Leu 180 185 190ttt cta cca aca tat gca caa gct
gca aat aca cat tta ttg cta tta 624Phe Leu Pro Thr Tyr Ala Gln Ala
Ala Asn Thr His Leu Leu Leu Leu 195 200 205aaa gat gct caa gtt ttt
gga gaa gaa tgg gga tat tct tca gaa gat 672Lys Asp Ala Gln Val Phe
Gly Glu Glu Trp Gly Tyr Ser Ser Glu Asp 210 215 220gtt gct gaa ttt
tat cat aga caa tta aaa ctt aca caa caa tac act 720Val Ala Glu Phe
Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr Thr225 230 235 240gac
cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga ggt 768Asp
His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg Gly 245 250
255tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa atg
816Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu Met
260 265 270act tta act gta tta gat cta att gta ctt ttc cca ttt tat
gat att 864Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr
Asp Ile 275 280 285cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca
aga gac att ttt 912Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr
Arg Asp Ile Phe 290 295 300acg gat cca att ttt tca ctt aat act ctt
cag gag tat gga cca act 960Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu
Gln Glu Tyr Gly Pro Thr305 310 315 320ttt ttg agt ata gaa aac tct
att cga aaa cct cat tta ttt gat tat 1008Phe Leu Ser Ile Glu Asn Ser
Ile Arg Lys Pro His Leu Phe Asp Tyr 325 330 335tta cag ggg att gaa
ttt cat acg cgt ctt caa cct ggt tac ttt ggg 1056Leu Gln Gly Ile Glu
Phe His Thr Arg Leu Gln Pro Gly Tyr Phe Gly 340 345 350aaa gat tct
ttc aat tat tgg tct ggt aat tat gta gaa act aga cct 1104Lys Asp Ser
Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg Pro 355 360 365agt
ata gga tct agt aag aca att act tcc cca ttt tat gga gat aaa 1152Ser
Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp Lys 370 375
380tct act gaa cct gta caa aag cta agc ttt gat gga caa aaa gtt tat
1200Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val
Tyr385 390 395 400cga act ata gct aat aca gac gta gcg gct tgg ccg
aat ggt aag gta 1248Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro
Asn Gly Lys Val 405 410 415tat tta ggt gtt acg aaa gtt gat ttt agt
caa tat gat gat caa aaa 1296Tyr Leu Gly Val Thr Lys Val Asp Phe Ser
Gln Tyr Asp Asp Gln Lys 420 425 430aat gaa act agt aca caa aca tat
gat tca aaa aga aac aat ggc cat 1344Asn Glu Thr Ser Thr Gln Thr Tyr
Asp Ser Lys Arg Asn Asn Gly His 435 440 445gta agt gca cag gat tct
att gac caa tta ccg cca gaa aca aca gat 1392Val Ser Ala Gln Asp Ser
Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp 450 455 460gaa cca ctt gaa
aaa gca tat agt cat cag ctt aat tac gcg gaa tgt 1440Glu Pro Leu Glu
Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu Cys465 470 475 480ttc
tta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg aca 1488Phe
Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp Thr 485 490
495cat aga agt gta gac ttt ttt aat aca att gat gct gaa aag att act
1536His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile Thr
500 505 510caa ctt cca gta gtg aaa gca tat gcc ttg tct tca ggt gct
tcc att 1584Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala
Ser Ile 515 520 525att gaa ggt cca gga ttc aca gga gga aat tta cta
ttc cta aaa gaa 1632Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu
Phe Leu Lys Glu 530 535 540tct agt aat tca att gct aaa ttt aaa gtt
aca tta aat tca gca gcc 1680Ser Ser Asn Ser Ile Ala Lys Phe Lys Val
Thr Leu Asn Ser Ala Ala545 550 555 560ttg tta caa cga tat cgt gta
aga ata cgc tat gct tct acc act aac 1728Leu Leu Gln Arg Tyr Arg Val
Arg Ile Arg Tyr Ala Ser Thr Thr Asn 565 570 575tta cga ctt ttt gtg
caa aat tca aac aat gat ttt ctt gtc atc tac 1776Leu Arg Leu Phe Val
Gln Asn Ser Asn Asn Asp Phe Leu Val Ile Tyr 580 585 590att aat aaa
act atg aat aaa gat gat gat tta aca tat caa aca ttt 1824Ile Asn Lys
Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr Phe 595 600 605gat
ctc gca act act aat tct aat atg ggg ttc tcg ggt gat aag aat 1872Asp
Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys Asn 610 615
620gaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaa atc tat
1920Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile
Tyr625 630 635 640ata gat aag ata gaa ttt atc cca gta caa ttg taa
1956Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
65052651PRTArtificial sequenceRecombinant delta endotoxin 52Met Asn
Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn
Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25
30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met
35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys
Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly
Val Val65 70 75 80Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr
Gln Ser Phe Leu 85 90 95Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys
Ala Phe Met Ala Gln 100 105 110Val Glu Val Leu Ile Asp Lys Lys Ile
Glu Glu Tyr Ala Lys Ser Lys 115 120 125Ala Leu Ala Glu Leu Gln Gly
Leu Gln Asn Asn Phe Glu Asp Tyr Val 130 135 140Asn Ala Leu Asn Ser
Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser Lys145 150 155 160Arg Ser
Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His 165 170
175Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val Leu
180 185 190Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu
Leu Leu 195 200 205Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr
Ser Ser Glu Asp 210 215 220Val Ala Glu Phe Tyr His Arg Gln Leu Lys
Leu Thr Gln Gln Tyr Thr225 230 235 240Asp His Cys Val Asn Trp Tyr
Asn Val Gly Leu Asn Gly Leu Arg Gly 245 250 255Ser Thr Tyr Asp Ala
Trp Val Lys Phe Asn Arg Phe Arg Arg Glu Met 260 265 270Thr Leu Thr
Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp Ile 275 280 285Arg
Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile Phe 290 295
300Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro
Thr305 310 315 320Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His
Leu Phe Asp Tyr 325 330 335Leu Gln Gly Ile Glu Phe His Thr Arg Leu
Gln Pro Gly Tyr Phe Gly 340 345 350Lys Asp Ser Phe Asn Tyr Trp Ser
Gly Asn Tyr Val Glu Thr Arg Pro 355 360 365Ser Ile Gly Ser Ser Lys
Thr Ile Thr Ser Pro Phe Tyr Gly Asp Lys 370 375 380Ser Thr Glu Pro
Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val Tyr385 390 395 400Arg
Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys Val 405 410
415Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln Lys
420 425 430Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn
Gly His 435 440 445Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro
Glu Thr Thr Asp 450 455 460Glu Pro Leu Glu Lys Ala Tyr Ser His Gln
Leu Asn Tyr Ala Glu Cys465 470 475 480Phe Leu Met Gln Asp Arg Arg
Gly Thr Ile Pro Phe Phe Thr Trp Thr 485 490 495His Arg Ser Val Asp
Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile Thr 500 505 510Gln Leu Pro
Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser Ile 515 520 525Ile
Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys Glu 530 535
540Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala
Ala545 550 555 560Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala
Ser Thr Thr Asn 565 570 575Leu Arg Leu Phe Val Gln Asn Ser Asn Asn
Asp Phe Leu Val Ile Tyr 580 585 590Ile Asn Lys Thr Met
Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr Phe 595 600 605Asp Leu Ala
Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys Asn 610 615 620Glu
Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile Tyr625 630
635 640Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
650531959DNAArtificial sequenceRecombinant delta endotoxin 53atg
aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met
Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10
15aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat
96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn
20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga
atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg
Met 35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta
aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val
Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att tta
ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu
Gly Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca ttt
tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe
Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gat gct gac cca
tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro
Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa gta ctg ata gat aag
aaa ata gag gag tat gct aaa agt 384Gln Val Glu Val Leu Ile Asp Lys
Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa gct ctt gca gag tta
cag ggt ctt caa aat aat ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu
Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140gtt aat gcg tta
aat tcc tgg aag aaa aca cct tta agt ttg cga agt 480Val Asn Ala Leu
Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160aaa
aga agc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt 528Lys
Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc gga ttc gaa gtg
576His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Gly Phe Glu Val
180 185 190ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta
ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga
tat tct tca gaa 672Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220gat gtt gct gaa ttt tat cat aga caa tta
aaa ctt aca caa caa tac 720Asp Val Ala Glu Phe Tyr His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240act gac cat tgt gtt aat tgg
tat aat gtt gga tta aat ggt tta aga 768Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act tat gat
gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg act tta
act gta tta gat cta att gta ctt ttc cca ttt tat gat 864Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285att
cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300ttt acg gat cca att ttt tca ctt aat act ctt cag gag tat gga cca
960Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly
Pro305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct
cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt
ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg
tct ggt aat tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt
aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa
cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400tat
cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa
1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac
aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445cat gta agt gca cag gat tct att gac caa tta ccg
cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat agt cat
cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag gac cgt
cgt gga aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta
gac ttt ttt aat aca att gat gct gaa aag att 1536Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt
cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att
att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca
1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560gcc ttg tta caa cga tat cgt gta aga ata cgc tat
gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca aac
aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590tac att aat aaa act atg aat aaa
gat gat gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act act
aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata
ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640tat
ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys
Ile Glu Phe Ile Pro Val Gln Leu 645 65054652PRTArtificial
sequenceRecombinant delta endotoxin 54Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg Ser Gln Asp Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Gly Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile Arg Leu Tyr Ser Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro
Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro305 310 315 320Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe
340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His
Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455
460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630 635 640Tyr Ile Asp Lys Ile Glu Phe
Ile Pro Val Gln Leu 645 650551956DNAArtificial sequenceRecombinant
delta endotoxin 55atg aat cca aac aat cga agt gaa cat gat acg ata
aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile
Lys Val Thr Pro1 5 10 15aac agt gaa ttg caa act aac cat aat caa tat
cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr
Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat
aaa gaa ttt tta aga atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr
Lys Glu Phe Leu Arg Met 35 40 45act gaa gac agt tct acg gaa gtg cta
gac aac tct aca gta aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu
Asp Asn Ser Thr Val Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt
gta ggg cag att tta ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val
Val Gly Gln Ile Leu Gly Val Val65 70 75 80gga gtt cca ttt gct ggg
gca ctc act tca ttt tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly
Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca
agt gaa gac cca tgg aag gct ttt atg gca caa 336Asn Thr Ile Trp Pro
Ser Glu Asp Pro Trp Lys Ala Phe Met Ala Gln 100 105 110gtt gaa gta
ctg ata gat aag aaa ata gag gag tat gct aaa agt aaa 384Val Glu Val
Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser Lys 115 120 125gct
ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat gtt 432Ala
Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr Val 130 135
140aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg cga aat cca
480Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Asn
Pro145 150 155 160cac agc caa ggt cga ata agg gaa ctt ttt tct caa
gca gaa agt cat 528His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln
Ala Glu Ser His 165 170 175ttt cgt aat tcc atg ccg tca ttt gca gtt
tcc aaa ttc gaa gtg ctg 576Phe Arg Asn Ser Met Pro Ser Phe Ala Val
Ser Lys Phe Glu Val Leu 180 185 190ttt cta cca aca tat gca caa gct
gca aat aca cat tta ttg cta tta 624Phe Leu Pro Thr Tyr Ala Gln Ala
Ala Asn Thr His Leu Leu Leu Leu 195 200 205aaa gat gct caa gtt ttt
gga gaa gaa tgg gga tat tct tca gaa gat 672Lys Asp Ala Gln Val Phe
Gly Glu Glu Trp Gly Tyr Ser Ser Glu Asp 210 215 220gtt gct gaa ttt
tat cat aga caa tta aaa ctt aca caa caa tac act 720Val Ala Glu Phe
Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr Thr225 230 235 240gac
cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga ggt 768Asp
His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg Gly 245 250
255tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa atg
816Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu Met
260 265 270act tta act gta tta gat cta att gta ctt ttc cca ttt tat
gat att 864Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr
Asp Ile 275 280 285cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca
aga gac att ttt 912Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr
Arg Asp Ile Phe 290 295 300acg gat cca att ttt tca ctt aat act ctt
cag gag tat gga cca act 960Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu
Gln Glu Tyr Gly Pro Thr305 310 315 320ttt ttg agt ata gaa aac tct
att cga aaa cct cat tta ttt gat tat 1008Phe Leu Ser Ile Glu Asn Ser
Ile Arg Lys Pro His Leu Phe Asp Tyr 325 330 335tta cag ggg att gaa
ttt cat acg cgt ctt caa cct ggt tac ttt ggg 1056Leu Gln Gly Ile Glu
Phe His Thr Arg Leu Gln Pro Gly Tyr Phe Gly 340 345 350aaa gat tct
ttc aat tat tgg tct ggt aat tat gta gaa act aga cct 1104Lys Asp Ser
Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg Pro 355 360 365agt
ata gga tct agt aag aca att act tcc cca ttt tat gga gat aaa 1152Ser
Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp Lys 370 375
380tct act gaa cct gta caa aag cta agc ttt gat gga caa aaa gtt tat
1200Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val
Tyr385 390 395 400cga act ata gct aat aca gac gta gcg gct tgg ccg
aat ggt aag gta 1248Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro
Asn Gly Lys Val 405 410 415tat tta ggt gtt acg aaa gtt gat ttt agt
caa tat gat gat caa aaa 1296Tyr Leu Gly Val Thr Lys Val Asp Phe Ser
Gln Tyr Asp Asp Gln Lys 420 425
430aat gaa act agt aca caa aca tat gat tca aaa aga aac aat ggc cat
1344Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly His
435 440 445gta agt gca cag gat tct att gac caa tta ccg cca gaa aca
aca gat 1392Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr
Thr Asp 450 455 460gaa cca ctt gaa aaa gca tat agt cat cag ctt aat
tac gcg gaa tgt 1440Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn
Tyr Ala Glu Cys465 470 475 480ttc tta atg cag gac cgt cgt gga aca
att cca ttt ttt act tgg aca 1488Phe Leu Met Gln Asp Arg Arg Gly Thr
Ile Pro Phe Phe Thr Trp Thr 485 490 495cat aga agt gta gac ttt ttt
aat aca att gat gct gaa aag att act 1536His Arg Ser Val Asp Phe Phe
Asn Thr Ile Asp Ala Glu Lys Ile Thr 500 505 510caa ctt cca gta gtg
aaa gca tat gcc ttg tct tca ggt gct tcc att 1584Gln Leu Pro Val Val
Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser Ile 515 520 525att gaa ggt
cca gga ttc aca gga gga aat tta cta ttc cta aaa gaa 1632Ile Glu Gly
Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys Glu 530 535 540tct
agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca gcc 1680Ser
Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala Ala545 550
555 560ttg tta caa cga tat cgt gta aga ata cgc tat gct tct acc act
aac 1728Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr
Asn 565 570 575tta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt
gtc atc tac 1776Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu
Val Ile Tyr 580 585 590att aat aaa act atg aat aaa gat gat gat tta
aca tat caa aca ttt 1824Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu
Thr Tyr Gln Thr Phe 595 600 605gat ctc gca act act aat tct aat atg
ggg ttc tcg ggt gat aag aat 1872Asp Leu Ala Thr Thr Asn Ser Asn Met
Gly Phe Ser Gly Asp Lys Asn 610 615 620gaa ctt ata ata gga gca gaa
tct ttc gtt tct aat gaa aaa atc tat 1920Glu Leu Ile Ile Gly Ala Glu
Ser Phe Val Ser Asn Glu Lys Ile Tyr625 630 635 640ata gat aag ata
gaa ttt atc cca gta caa ttg taa 1956Ile Asp Lys Ile Glu Phe Ile Pro
Val Gln Leu 645 65056651PRTArtificial sequenceRecombinant delta
endotoxin 56Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val
Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu
Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu
Phe Leu Arg Met 35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn
Ser Thr Val Lys Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val Val Gly
Gln Ile Leu Gly Val Val65 70 75 80Gly Val Pro Phe Ala Gly Ala Leu
Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95Asn Thr Ile Trp Pro Ser Glu
Asp Pro Trp Lys Ala Phe Met Ala Gln 100 105 110Val Glu Val Leu Ile
Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser Lys 115 120 125Ala Leu Ala
Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr Val 130 135 140Asn
Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Asn Pro145 150
155 160His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser
His 165 170 175Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe
Glu Val Leu 180 185 190Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr
His Leu Leu Leu Leu 195 200 205Lys Asp Ala Gln Val Phe Gly Glu Glu
Trp Gly Tyr Ser Ser Glu Asp 210 215 220Val Ala Glu Phe Tyr His Arg
Gln Leu Lys Leu Thr Gln Gln Tyr Thr225 230 235 240Asp His Cys Val
Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg Gly 245 250 255Ser Thr
Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu Met 260 265
270Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp Ile
275 280 285Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp
Ile Phe 290 295 300Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu
Tyr Gly Pro Thr305 310 315 320Phe Leu Ser Ile Glu Asn Ser Ile Arg
Lys Pro His Leu Phe Asp Tyr 325 330 335Leu Gln Gly Ile Glu Phe His
Thr Arg Leu Gln Pro Gly Tyr Phe Gly 340 345 350Lys Asp Ser Phe Asn
Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg Pro 355 360 365Ser Ile Gly
Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp Lys 370 375 380Ser
Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val Tyr385 390
395 400Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys
Val 405 410 415Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp
Asp Gln Lys 420 425 430Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys
Arg Asn Asn Gly His 435 440 445Val Ser Ala Gln Asp Ser Ile Asp Gln
Leu Pro Pro Glu Thr Thr Asp 450 455 460Glu Pro Leu Glu Lys Ala Tyr
Ser His Gln Leu Asn Tyr Ala Glu Cys465 470 475 480Phe Leu Met Gln
Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp Thr 485 490 495His Arg
Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile Thr 500 505
510Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser Ile
515 520 525Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu
Lys Glu 530 535 540Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu
Asn Ser Ala Ala545 550 555 560Leu Leu Gln Arg Tyr Arg Val Arg Ile
Arg Tyr Ala Ser Thr Thr Asn 565 570 575Leu Arg Leu Phe Val Gln Asn
Ser Asn Asn Asp Phe Leu Val Ile Tyr 580 585 590Ile Asn Lys Thr Met
Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr Phe 595 600 605Asp Leu Ala
Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys Asn 610 615 620Glu
Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile Tyr625 630
635 640Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
650571956DNAArtificial sequenceRecombinant delta endotoxin 57atg
aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met
Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10
15aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat
96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn
20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga
atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg
Met 35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta
aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val
Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att tta
ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu
Gly Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca ttt
tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe
Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gaa gac cca tgg
aag gct ttt atg gca caa 336Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp
Lys Ala Phe Met Ala Gln 100 105 110gtt gaa gta ctg ata gat aag aaa
ata gag gag tat gct aaa agt aaa 384Val Glu Val Leu Ile Asp Lys Lys
Ile Glu Glu Tyr Ala Lys Ser Lys 115 120 125gct ctt gca gag tta cag
ggt ctt caa aat aat ttc gaa gat tat gtt 432Ala Leu Ala Glu Leu Gln
Gly Leu Gln Asn Asn Phe Glu Asp Tyr Val 130 135 140aat gcg tta aat
tcc tgg aag aaa ttt cac cat tct cgt cgt tct aaa 480Asn Ala Leu Asn
Ser Trp Lys Lys Phe His His Ser Arg Arg Ser Lys145 150 155 160aga
agc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt cat 528Arg
Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His 165 170
175ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg ctg
576Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val Leu
180 185 190ttt cta cca aca tat gca caa gct gca aat aca cat tta ttg
cta tta 624Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu
Leu Leu 195 200 205aaa gat gct caa gtt ttt gga gaa gaa tgg gga tat
tct tca gaa gat 672Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr
Ser Ser Glu Asp 210 215 220gtt gct gaa ttt tat cat aga caa tta aaa
ctt aca caa caa tac act 720Val Ala Glu Phe Tyr His Arg Gln Leu Lys
Leu Thr Gln Gln Tyr Thr225 230 235 240gac cat tgt gtt aat tgg tat
aat gtt gga tta aat ggt tta aga ggt 768Asp His Cys Val Asn Trp Tyr
Asn Val Gly Leu Asn Gly Leu Arg Gly 245 250 255tca act tat gat gca
tgg gtc aaa ttt aac cgt ttt cgc aga gaa atg 816Ser Thr Tyr Asp Ala
Trp Val Lys Phe Asn Arg Phe Arg Arg Glu Met 260 265 270act tta act
gta tta gat cta att gta ctt ttc cca ttt tat gat att 864Thr Leu Thr
Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp Ile 275 280 285cgg
tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att ttt 912Arg
Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile Phe 290 295
300acg gat cca att ttt tca ctt aat act ctt cag gag tat gga cca act
960Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro
Thr305 310 315 320ttt ttg agt ata gaa aac tct att cga aaa cct cat
tta ttt gat tat 1008Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His
Leu Phe Asp Tyr 325 330 335tta cag ggg att gaa ttt cat acg cgt ctt
caa cct ggt tac ttt ggg 1056Leu Gln Gly Ile Glu Phe His Thr Arg Leu
Gln Pro Gly Tyr Phe Gly 340 345 350aaa gat tct ttc aat tat tgg tct
ggt aat tat gta gaa act aga cct 1104Lys Asp Ser Phe Asn Tyr Trp Ser
Gly Asn Tyr Val Glu Thr Arg Pro 355 360 365agt ata gga tct agt aag
aca att act tcc cca ttt tat gga gat aaa 1152Ser Ile Gly Ser Ser Lys
Thr Ile Thr Ser Pro Phe Tyr Gly Asp Lys 370 375 380tct act gaa cct
gta caa aag cta agc ttt gat gga caa aaa gtt tat 1200Ser Thr Glu Pro
Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val Tyr385 390 395 400cga
act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag gta 1248Arg
Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys Val 405 410
415tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa aaa
1296Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln Lys
420 425 430aat gaa act agt aca caa aca tat gat tca aaa aga aac aat
ggc cat 1344Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn
Gly His 435 440 445gta agt gca cag gat tct att gac caa tta ccg cca
gaa aca aca gat 1392Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro
Glu Thr Thr Asp 450 455 460gaa cca ctt gaa aaa gca tat agt cat cag
ctt aat tac gcg gaa tgt 1440Glu Pro Leu Glu Lys Ala Tyr Ser His Gln
Leu Asn Tyr Ala Glu Cys465 470 475 480ttc tta atg cag gac cgt cgt
gga aca att cca ttt ttt act tgg aca 1488Phe Leu Met Gln Asp Arg Arg
Gly Thr Ile Pro Phe Phe Thr Trp Thr 485 490 495cat aga agt gta gac
ttt ttt aat aca att gat gct gaa aag att act 1536His Arg Ser Val Asp
Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile Thr 500 505 510caa ctt cca
gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc att 1584Gln Leu Pro
Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser Ile 515 520 525att
gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa gaa 1632Ile
Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys Glu 530 535
540tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca gcc
1680Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala
Ala545 550 555 560ttg tta caa cga tat cgt gta aga ata cgc tat gct
tct acc act aac 1728Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala
Ser Thr Thr Asn 565 570 575tta cga ctt ttt gtg caa aat tca aac aat
gat ttt ctt gtc atc tac 1776Leu Arg Leu Phe Val Gln Asn Ser Asn Asn
Asp Phe Leu Val Ile Tyr 580 585 590att aat aaa act atg aat aaa gat
gat gat tta aca tat caa aca ttt 1824Ile Asn Lys Thr Met Asn Lys Asp
Asp Asp Leu Thr Tyr Gln Thr Phe 595 600 605gat ctc gca act act aat
tct aat atg ggg ttc tcg ggt gat aag aat 1872Asp Leu Ala Thr Thr Asn
Ser Asn Met Gly Phe Ser Gly Asp Lys Asn 610 615 620gaa ctt ata ata
gga gca gaa tct ttc gtt tct aat gaa aaa atc tat 1920Glu Leu Ile Ile
Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile Tyr625 630 635 640ata
gat aag ata gaa ttt atc cca gta caa ttg taa 1956Ile Asp Lys Ile Glu
Phe Ile Pro Val Gln Leu 645 65058651PRTArtificial
sequenceRecombinant delta endotoxin 58Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Ala Gln
100 105 110Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys
Ser Lys 115 120 125Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe
Glu Asp Tyr Val 130 135 140Asn Ala Leu Asn Ser Trp Lys Lys Phe His
His Ser Arg Arg Ser Lys145 150 155 160Arg Ser Gln Asp Arg Ile Arg
Glu Leu Phe Ser Gln Ala Glu Ser His 165 170 175Phe Arg Asn Ser Met
Pro Ser Phe Ala Val Ser Lys Phe Glu Val Leu 180 185 190Phe Leu Pro
Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu Leu 195 200 205Lys
Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu Asp 210 215
220Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr
Thr225 230 235 240Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn
Gly Leu Arg Gly 245 250 255Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn
Arg Phe Arg Arg Glu Met 260 265 270Thr Leu Thr Val Leu Asp Leu Ile
Val Leu Phe Pro Phe Tyr Asp Ile 275 280 285Arg Leu Tyr Ser Lys Gly
Val Lys Thr Glu Leu Thr Arg Asp Ile Phe 290 295 300Thr Asp Pro Ile
Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro Thr305 310 315 320Phe
Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp Tyr 325 330
335Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe Gly
340 345 350Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr
Arg Pro 355 360 365Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe
Tyr Gly Asp Lys 370 375 380Ser Thr Glu Pro Val Gln Lys Leu Ser Phe
Asp Gly Gln Lys Val Tyr385 390 395
400Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys Val
405 410 415Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp
Gln Lys 420 425 430Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg
Asn Asn Gly His 435 440 445Val Ser Ala Gln Asp Ser Ile Asp Gln Leu
Pro Pro Glu Thr Thr Asp 450 455 460Glu Pro Leu Glu Lys Ala Tyr Ser
His Gln Leu Asn Tyr Ala Glu Cys465 470 475 480Phe Leu Met Gln Asp
Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp Thr 485 490 495His Arg Ser
Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile Thr 500 505 510Gln
Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser Ile 515 520
525Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys Glu
530 535 540Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala Ala545 550 555 560Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr Asn 565 570 575Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile Tyr 580 585 590Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr Phe 595 600 605Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys Asn 610 615 620Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile Tyr625 630 635
640Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
650591959DNAArtificial sequenceRecombinant delta endotoxin 59atg
aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met
Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10
15aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat
96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn
20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga
atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg
Met 35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta
aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val
Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att tta
ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu
Gly Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca ttt
tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe
Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gat gct gac cca
tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro
Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa gta ctg ata gat aag
aaa ata gag gag tat gct aaa agt 384Gln Val Glu Val Leu Ile Asp Lys
Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa gct ctt gca gag tta
cag ggt ctt caa aat aat ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu
Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140gtt aat gcg tta
aat tcc tgg aag aaa aca cct tta agt ttg cga agt 480Val Asn Ala Leu
Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160aaa
aga agc caa ggt cga ata agg gaa ctt ttt tct caa gca gaa agt 528Lys
Arg Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg
576His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta
ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga
tat tct tca gaa 672Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220gat gtt gct gaa ttt tat cat aga caa tta
aaa ctt aca caa caa tac 720Asp Val Ala Glu Phe Tyr His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240act gac cat tgt gtt aat tgg
tat aat gtt gga tta aat ggt tta aga 768Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act tat gat
gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg act tta
act gta tta gat cta att gta ctt ttc cca ttt tat gat 864Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285att
cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300ttt acg gat cca att ttt acc ctt aat aca cta cag aag tac gga cca
960Phe Thr Asp Pro Ile Phe Thr Leu Asn Thr Leu Gln Lys Tyr Gly
Pro305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct
cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt
ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg
tct ggt aat tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt
aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa
cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400tat
cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa
1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac
aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445cat gta agt gca cag gat tct att gac caa tta ccg
cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat agt cat
cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag gac cgt
cgt gga aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta
gac ttt ttt aat aca att gat gct gaa aag att 1536Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt
cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att
att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca
1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560gcc ttg tta caa cga tat cgt gta aga ata cgc tat
gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca aac
aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590tac att aat aaa act atg aat aaa
gat gat gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act act
aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata
ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640tat
ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys
Ile Glu Phe Ile Pro Val Gln Leu 645 65060652PRTArtificial
sequenceRecombinant delta endotoxin 60Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg Ser Gln Gly Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile Arg Leu Tyr Ser Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro
Ile Phe Thr Leu Asn Thr Leu Gln Lys Tyr Gly Pro305 310 315 320Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe
340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His
Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455
460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630 635 640Tyr Ile Asp Lys Ile Glu Phe
Ile Pro Val Gln Leu 645 650611959DNAArtificial sequenceRecombinant
delta endotoxin 61atg aat cca aac aat cga agt gaa cat gat acg ata
aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile
Lys Val Thr Pro1 5 10 15aac agt gaa ttg caa act aac cat aat caa tat
cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr
Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat
aaa gaa ttt tta aga atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr
Lys Glu Phe Leu Arg Met 35 40 45act gaa gac agt tct acg gaa gtg cta
gac aac tct aca gta aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu
Asp Asn Ser Thr Val Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt
gta ggg cag att tta ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val
Val Gly Gln Ile Leu Gly Val Val65 70 75 80gga gtt cca ttt gct ggg
gca ctc act tca ttt tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly
Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca
agt gat gct gac cca tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro
Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa
gta ctg ata gat aag aaa ata gag gag tat gct aaa agt 384Gln Val Glu
Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa
gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat 432Lys
Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135
140gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg cga agt
480Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg
Ser145 150 155 160aaa aga agc caa ggt cga ata agg gaa ctt ttt tct
caa gca gaa agt 528Lys Arg Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser
Gln Ala Glu Ser 165 170 175cat ttt cgt aat tcc atg ccg tca ttt gca
gtt tcc aaa ttc gaa gtg 576His Phe Arg Asn Ser Met Pro Ser Phe Ala
Val Ser Lys Phe Glu Val 180 185 190ctg ttt cta cca aca tat gca caa
gct gca aat aca cat tta ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln
Ala Ala Asn Thr His Leu Leu Leu 195 200 205tta aaa gat gct caa gtt
ttt gga gaa gaa tgg gga tat tct tca gaa 672Leu Lys Asp Ala Gln Val
Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220gat gtt gct gaa
ttt tat cat aga caa tta aaa ctt aca caa caa tac 720Asp Val Ala Glu
Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240act
gac cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga 768Thr
Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250
255ggt tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa
816Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu
260 265 270atg act tta act gta tta gat cta att gta ctt ttc cca ttt
tat gat 864Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe
Tyr Asp 275 280 285gtt cgg tta tac cca aaa ggg gtt aaa aca gaa cta
aca aga gac att 912Val Arg Leu Tyr Pro Lys Gly Val Lys Thr Glu Leu
Thr Arg Asp Ile 290 295 300tct acg gat cca att ttt gcc gtt aat act
ctg tgg gaa tac gga cca
960Ser Thr Asp Pro Ile Phe Ala Val Asn Thr Leu Trp Glu Tyr Gly
Pro305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct
cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt
ctt cga cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Arg Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg
tct ggt aat tat gca gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Ala Glu Thr Arg 355 360 365cct agt ata gga tct agt
aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa
cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400tat
cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa
1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac
aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445cat gta agt gca cag gat tct att gac caa tta ccg
cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat agt cat
cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag gac cgt
cgt gga aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta
gac ttt ttt aat aca att gat gct gaa aag att 1536Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt
cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att
att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca
1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560gcc ttg tta caa cga tat cgt gta aga ata cgc tat
gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca aac
aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590tac att aat aaa act atg aat aaa
gat gat gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act act
aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata
ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640tat
ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys
Ile Glu Phe Ile Pro Val Gln Leu 645 65062652PRTArtificial
sequenceRecombinant delta endotoxin 62Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg Ser Gln Gly Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Val Arg Leu Tyr Pro Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Ser Thr Asp Pro
Ile Phe Ala Val Asn Thr Leu Trp Glu Tyr Gly Pro305 310 315 320Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Arg Pro Gly Tyr Phe
340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Ala Glu
Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His
Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455
460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630 635 640Tyr Ile Asp Lys Ile Glu Phe
Ile Pro Val Gln Leu 645 650631959DNAArtificial sequenceRecombinant
delta endotoxin 63atg aat cca aac aat cga agt gaa cat gat acg ata
aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile
Lys Val Thr Pro1 5 10 15aac agt gaa ttg caa act aac cat aat caa tat
cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr
Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat
aaa gaa ttt tta aga atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr
Lys Glu Phe Leu Arg Met 35 40 45act gaa gac agt tct acg gaa gtg cta
gac aac tct aca gta aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu
Asp Asn Ser Thr Val Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt
gta ggg cag att tta ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val
Val Gly Gln Ile Leu Gly Val Val65 70 75 80gga gtt cca ttt gct ggg
gca ctc act tca ttt tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly
Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca
agt gat gct gac cca tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro
Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa
gta ctg ata gat aag aaa ata gag gag tat gct aaa agt 384Gln Val Glu
Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa
gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat 432Lys
Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135
140gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg cga agt
480Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg
Ser145 150 155 160aaa aga agc caa gat cga ata agg gaa ctt ttt tct
caa gca gaa agt 528Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser
Gln Ala Glu Ser 165 170 175cat ttt cgt aat tcc atg ccg tca ttt gca
gtt tcc aaa ttc gaa gtg 576His Phe Arg Asn Ser Met Pro Ser Phe Ala
Val Ser Lys Phe Glu Val 180 185 190ctg ttt cta cca aca tat gca caa
gct gca aat aca cat tta ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln
Ala Ala Asn Thr His Leu Leu Leu 195 200 205tta aaa gat gct caa gtt
ttt gga gaa gaa tgg gga tat tct tca gaa 672Leu Lys Asp Ala Gln Val
Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220gat gtt gct gaa
ttt tat cat aga caa tta aaa ctt aca caa caa tac 720Asp Val Ala Glu
Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240act
gac cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga 768Thr
Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250
255ggt tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa
816Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu
260 265 270atg act tta act gta tta gat cta att gta ctt ttc cca ttt
tat gat 864Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe
Tyr Asp 275 280 285gtt cgg tta tac cca aaa ggg gtt aaa aca gaa cta
aca aga gac att 912Val Arg Leu Tyr Pro Lys Gly Val Lys Thr Glu Leu
Thr Arg Asp Ile 290 295 300ttt acg gat cca att ttt tca ctt aat act
ctt cag gag tat gga cca 960Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr
Leu Gln Glu Tyr Gly Pro305 310 315 320act ttt ttg agt ata gaa aac
tct att cga aaa cct cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn
Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335tat tta cag ggg att
gaa ttt cat acg cgt ctt cga cct ggt tac ttt 1056Tyr Leu Gln Gly Ile
Glu Phe His Thr Arg Leu Arg Pro Gly Tyr Phe 340 345 350ggg aaa gat
tct ttc aat tat tgg tct ggt aat tat gta gaa act aga 1104Gly Lys Asp
Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct
agt ata gga tct agt aag aca att act tcc cca ttt tat gga gat 1152Pro
Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375
380aaa tct act gaa cct gta caa aag cta agc ttt gat gga caa aaa gtt
1200Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys
Val385 390 395 400tat cga act ata gct aat aca gac gta gcg gct tgg
ccg aat ggt aag 1248Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp
Pro Asn Gly Lys 405 410 415gta tat tta ggt gtt acg aaa gtt gat ttt
agt caa tat gat gat caa 1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe
Ser Gln Tyr Asp Asp Gln 420 425 430aaa aat gaa act agt aca caa aca
tat gat tca aaa aga aac aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr
Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445cat gta agt gca cag gat
tct att gac caa tta ccg cca gaa aca aca 1392His Val Ser Ala Gln Asp
Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460gat gaa cca ctt
gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu
Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480tgt
ttc tta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg 1488Cys
Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490
495aca cat aga agt gta gac ttt ttt aat aca att gat gct gaa aag att
1536Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile
500 505 510act caa ctt cca gta gtg aaa gca tat gcc ttg tct tca ggt
gct tcc 1584Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly
Ala Ser 515 520 525att att gaa ggt cca gga ttc aca gga gga aat tta
cta ttc cta aaa 1632Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu
Leu Phe Leu Lys 530 535 540gaa tct agt aat tca att gct aaa ttt aaa
gtt aca tta aat tca gca 1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys
Val Thr Leu Asn Ser Ala545 550 555 560gcc ttg tta caa cga tat cgt
gta aga ata cgc tat gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg
Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt
gtg caa aat tca aac aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe
Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590tac att aat
aaa act atg aat aaa gat gat gat tta aca tat caa aca 1824Tyr Ile Asn
Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt
gat ctc gca act act aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe
Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615
620aat gaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaa atc
1920Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys
Ile625 630 635 640tat ata gat aag ata gaa ttt atc cca gta caa ttg
taa 1959Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
65064652PRTArtificial sequenceRecombinant delta endotoxin 64Met Asn
Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn
Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25
30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met
35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys
Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly
Val Val65 70 75 80Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr
Gln Ser Phe Leu 85 90 95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp
Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys
Ile Glu Glu Tyr Ala Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln
Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn
Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg
Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His
Leu
Leu Leu 195 200 205Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220Asp Val Ala Glu Phe Tyr His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Val
Arg Leu Tyr Pro Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly
Pro305 310 315 320Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Arg Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640Tyr
Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
650651959DNAArtificial sequenceRecombinant delta endotoxin 65atg
aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met
Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10
15aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat
96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn
20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga
atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg
Met 35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta
aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val
Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt gta ggg cag att tta
ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu
Gly Val Val65 70 75 80gga gtt cca ttt gct ggg gca ctc act tca ttt
tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe
Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca agt gat gct gac cca
tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro
Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa gta ctg ata gat aag
aaa ata gag gag tat gct aaa agt 384Gln Val Glu Val Leu Ile Asp Lys
Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa gct ctt gca gag tta
cag ggt ctt caa aat aat ttc gaa gat tat 432Lys Ala Leu Ala Glu Leu
Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140gtt aat gcg tta
aat tcc tgg aag aaa aca cct tta agt ttg cga agt 480Val Asn Ala Leu
Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160aaa
aga agc caa ggt cga ata agg gaa ctt ttt tct caa gca gaa agt 528Lys
Arg Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg
576His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta
ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga
tat tct tca gaa 672Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220gat gtt gct gaa ttt tat cat aga caa tta
aaa ctt aca caa caa tac 720Asp Val Ala Glu Phe Tyr His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240act gac cat tgt gtt aat tgg
tat aat gtt gga tta aat ggt tta aga 768Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act tat gat
gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg act tta
act gta tta gat cta att gta ctt ttc cca ttt tat gat 864Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285att
cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300ttt acg gat cca att ttt tta ctt aat act ctt cag gag tat gga cca
960Phe Thr Asp Pro Ile Phe Leu Leu Asn Thr Leu Gln Glu Tyr Gly
Pro305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct
cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt
ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg
tct ggt aat tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt
aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa
cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400tat
cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa
1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac
aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445cat gta agt gca cag gat tct att gac caa tta ccg
cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460gat gaa cca ctt gaa aaa gca tat agt cat
cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480tgt ttc tta atg cag gac cgt
cgt gga aca att cca ttt ttt act tgg 1488Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta
gac ttt ttt aat aca att gat gct gaa aag att 1536Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt
cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att
att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca
1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560gcc ttg tta caa cga tat cgt gta aga ata cgc tat
gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt gtg caa aat tca aac
aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590tac att aat aaa act atg aat aaa
gat gat gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt gat ctc gca act act
aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620aat gaa ctt ata
ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640tat
ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys
Ile Glu Phe Ile Pro Val Gln Leu 645 65066652PRTArtificial
sequenceRecombinant delta endotoxin 66Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg Ser Gln Gly Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile Arg Leu Tyr Ser Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro
Ile Phe Leu Leu Asn Thr Leu Gln Glu Tyr Gly Pro305 310 315 320Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe
340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His
Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455
460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630 635 640Tyr Ile Asp Lys Ile Glu Phe
Ile Pro Val Gln Leu 645 650671959DNAArtificial sequenceRecombinant
delta endotoxin 67atg aat cca aac aat cga agt gaa cat gat acg ata
aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile
Lys Val Thr Pro1 5 10 15aac agt gaa ttg caa act aac cat aat caa tat
cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr
Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat
aaa gaa ttt tta aga atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr
Lys Glu Phe Leu Arg Met 35 40 45act gaa gac agt tct acg gaa gtg cta
gac aac tct aca gta aaa gat 192Thr Glu Asp Ser Ser Thr Glu Val Leu
Asp Asn Ser Thr Val Lys Asp 50 55 60gca gtt ggg aca gga att tct gtt
gta ggg cag att tta ggt gtt gta 240Ala Val Gly Thr Gly Ile Ser Val
Val Gly Gln Ile Leu Gly Val Val65 70 75 80gga gtt cca ttt gct ggg
gca ctc act tca ttt tat caa tca ttt ctt 288Gly Val Pro Phe Ala Gly
Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95aac act ata tgg cca
agt gat gct gac cca tgg aag gct ttt atg gca 336Asn Thr Ile Trp Pro
Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110caa gtt gaa
gta ctg ata gat aag aaa ata gag gag tat gct aaa agt 384Gln Val Glu
Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125aaa
gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat 432Lys
Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135
140gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg cga agt
480Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg
Ser145 150 155 160aaa aga agc caa gat cga ata agg gaa ctt ttt tct
caa gca gaa agt 528Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser
Gln Ala Glu Ser 165 170 175cat ttt cgt aat tcc atg ccg tca ttt gca
gtt tcc aaa ttc gaa gtg 576His Phe Arg Asn Ser Met Pro Ser Phe
Ala
Val Ser Lys Phe Glu Val 180 185 190ctg ttt cta cca aca tat gca caa
gct gca aat aca cat tta ttg cta 624Leu Phe Leu Pro Thr Tyr Ala Gln
Ala Ala Asn Thr His Leu Leu Leu 195 200 205tta aaa gat gct caa gtt
ttt gga gaa gaa tgg gga tat tct tca gaa 672Leu Lys Asp Ala Gln Val
Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220gat gtt gct gaa
ttt tat cat aga caa tta aaa ctt aca caa caa tac 720Asp Val Ala Glu
Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240act
gac cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga 768Thr
Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250
255ggt tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa
816Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu
260 265 270atg act tta act gta tta gat cta att gta ctt ttc cca ttt
tat gat 864Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe
Tyr Asp 275 280 285att cgg tta tac tca aaa ggg gtt aaa aca gaa cta
aca aga gac att 912Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu
Thr Arg Asp Ile 290 295 300ttt acg gat cca att ttt tca ctt aat act
ctt cag gag tat gga cca 960Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr
Leu Gln Glu Tyr Gly Pro305 310 315 320act ttt ttg agt ata gaa aac
tct att cga aaa cct cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn
Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335tat tta cag ggg att
gaa ttt cat acg cgt ctt cga cct ggt tac ttt 1056Tyr Leu Gln Gly Ile
Glu Phe His Thr Arg Leu Arg Pro Gly Tyr Phe 340 345 350ggg aaa gat
tct ttc aat tat tgg tct ggt aat tat gta gaa act aga 1104Gly Lys Asp
Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct
agt ata gga tct agt aag aca att act tcc cca ttt tat gga gat 1152Pro
Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375
380aaa tct act gaa cct gta caa aag cta agc ttt gat gga caa aaa gtt
1200Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys
Val385 390 395 400tat cga act ata gct aat aca gac gta gcg gct tgg
ccg aat ggt aag 1248Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp
Pro Asn Gly Lys 405 410 415gta tat tta ggt gtt acg aaa gtt gat ttt
agt caa tat gat gat caa 1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe
Ser Gln Tyr Asp Asp Gln 420 425 430aaa aat gaa act agt aca caa aca
tat gat tca aaa aga aac aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr
Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445cat gta agt gca cag gat
tct att gac caa tta ccg cca gaa aca aca 1392His Val Ser Ala Gln Asp
Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460gat gaa cca ctt
gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu
Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480tgt
ttc tta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg 1488Cys
Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490
495aca cat aga agt gta gac ttt ttt aat aca att gat gct gaa aag att
1536Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile
500 505 510act caa ctt cca gta gtg aaa gca tat gcc ttg tct tca ggt
gct tcc 1584Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly
Ala Ser 515 520 525att att gaa ggt cca gga ttc aca gga gga aat tta
cta ttc cta aaa 1632Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu
Leu Phe Leu Lys 530 535 540gaa tct agt aat tca att gct aaa ttt aaa
gtt aca tta aat tca gca 1680Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys
Val Thr Leu Asn Ser Ala545 550 555 560gcc ttg tta caa cga tat cgt
gta aga ata cgc tat gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg
Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575aac tta cga ctt ttt
gtg caa aat tca aac aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe
Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590tac att aat
aaa act atg aat aaa gat gat gat tta aca tat caa aca 1824Tyr Ile Asn
Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605ttt
gat ctc gca act act aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe
Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615
620aat gaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaa atc
1920Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys
Ile625 630 635 640tat ata gat aag ata gaa ttt atc cca gta caa ttg
taa 1959Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
65068652PRTArtificial sequenceRecombinant delta endotoxin 68Met Asn
Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn
Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25
30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met
35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys
Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly
Val Val65 70 75 80Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr
Gln Ser Phe Leu 85 90 95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp
Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys
Ile Glu Glu Tyr Ala Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln
Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn
Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg
Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170
175His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val
180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu Leu 195 200 205Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly
Tyr Ser Ser Glu 210 215 220Asp Val Ala Glu Phe Tyr His Arg Gln Leu
Lys Leu Thr Gln Gln Tyr225 230 235 240Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp
Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu
Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295
300Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly
Pro305 310 315 320Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro
His Leu Phe Asp 325 330 335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
Leu Arg Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser
Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu
Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400Tyr
Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410
415Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln
420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Asn Gly 435 440 445His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr 450 455 460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
Gln Leu Asn Tyr Ala Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg
Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val
Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu
Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535
540Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser
Ala545 550 555 560Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr 565 570 575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr Met Asn Lys
Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr
Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile
Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640Tyr
Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
650691482DNAArtificial sequenceRecombinant delta endotoxin 69agt
aaa aga agc caa gat cga ata agg gaa ctt ttt tct caa gca gaa 48Ser
Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu1 5 10
15agt cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa
96Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu
20 25 30gtg ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta
ttg 144Val Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Leu 35 40 45cta tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga tat
tct tca 192Leu Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr
Ser Ser 50 55 60gaa gat gtt gct gaa ttt tat cat aga caa tta aaa ctt
aca caa caa 240Glu Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu
Thr Gln Gln65 70 75 80tac act gac cat tgt gtt aat tgg tat aat gtt
gga tta aat ggt tta 288Tyr Thr Asp His Cys Val Asn Trp Tyr Asn Val
Gly Leu Asn Gly Leu 85 90 95aga ggt tca act tat gat gca tgg gtc aaa
ttt aac cgt ttt cgc aga 336Arg Gly Ser Thr Tyr Asp Ala Trp Val Lys
Phe Asn Arg Phe Arg Arg 100 105 110gaa atg act tta act gta tta gat
cta att gta ctt ttc cca ttt tat 384Glu Met Thr Leu Thr Val Leu Asp
Leu Ile Val Leu Phe Pro Phe Tyr 115 120 125gat att cgg tta tac tca
aaa ggg gtt aaa aca gaa cta aca aga gac 432Asp Ile Arg Leu Tyr Ser
Lys Gly Val Lys Thr Glu Leu Thr Arg Asp 130 135 140att ttt acg gat
cca att ttt tca ctt aat act ctt cag gag tat gga 480Ile Phe Thr Asp
Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly145 150 155 160cca
act ttt ttg agt ata gaa aac tct att cga aaa cct cat tta ttt 528Pro
Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe 165 170
175gat tat tta cag ggg att gaa ttt cat acg cgt ctt caa cct ggt tac
576Asp Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr
180 185 190ttt ggg aaa gat tct ttc aat tat tgg tct ggt aat tat gta
gaa act 624Phe Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val
Glu Thr 195 200 205aga cct agt ata gga tct agt aag aca att act tcc
cca ttt tat gga 672Arg Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser
Pro Phe Tyr Gly 210 215 220gat aaa tct act gaa cct gta caa aag cta
agc ttt gat gga caa aaa 720Asp Lys Ser Thr Glu Pro Val Gln Lys Leu
Ser Phe Asp Gly Gln Lys225 230 235 240gtt tat cga act ata gct aat
aca gac gta gcg gct tgg ccg aat ggt 768Val Tyr Arg Thr Ile Ala Asn
Thr Asp Val Ala Ala Trp Pro Asn Gly 245 250 255aag gta tat tta ggt
gtt acg aaa gtt gat ttt agt caa tat gat gat 816Lys Val Tyr Leu Gly
Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp 260 265 270caa aaa aat
gaa act agt aca caa aca tat gat tca aaa aga aac aat 864Gln Lys Asn
Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn 275 280 285ggc
cat gta agt gca cag gat tct att gac caa tta ccg cca gaa aca 912Gly
His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr 290 295
300aca gat gaa cca ctt gaa aaa gca tat agt cat cag ctt aat tac gcg
960Thr Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr
Ala305 310 315 320gaa tgt ttc tta atg cag gac cgt cgt gga aca att
cca ttt ttt act 1008Glu Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile
Pro Phe Phe Thr 325 330 335tgg aca cat aga agt gta gac ttt ttt aat
aca att gat gct gaa aag 1056Trp Thr His Arg Ser Val Asp Phe Phe Asn
Thr Ile Asp Ala Glu Lys 340 345 350att act caa ctt cca gta gtg aaa
gca tat gcc ttg tct tca ggt gct 1104Ile Thr Gln Leu Pro Val Val Lys
Ala Tyr Ala Leu Ser Ser Gly Ala 355 360 365tcc att att gaa ggt cca
gga ttc aca gga gga aat tta cta ttc cta 1152Ser Ile Ile Glu Gly Pro
Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu 370 375 380aaa gaa tct agt
aat tca att gct aaa ttt aaa gtt aca tta aat tca 1200Lys Glu Ser Ser
Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser385 390 395 400gca
gcc ttg tta caa cga tat cgt gta aga ata cgc tat gct tct acc 1248Ala
Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr 405 410
415act aac tta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtc
1296Thr Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val
420 425 430atc tac att aat aaa act atg aat aaa gat gat gat tta aca
tat caa 1344Ile Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr
Tyr Gln 435 440 445aca ttt gat ctc gca act act aat tct aat atg ggg
ttc tcg ggt gat 1392Thr Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly
Phe Ser Gly Asp 450 455 460aag aat gaa ctt ata ata gga gca gaa tct
ttc gtt tct aat gaa aaa 1440Lys Asn Glu Leu Ile Ile Gly Ala Glu Ser
Phe Val Ser Asn Glu Lys465 470 475 480atc tat ata gat aag ata gaa
ttt atc cca gta caa ttg taa 1482Ile Tyr Ile Asp Lys Ile Glu Phe Ile
Pro Val Gln Leu 485 49070493PRTArtificial sequenceRecombinant delta
endotoxin 70Ser Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln
Ala Glu1 5 10 15Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser
Lys Phe Glu 20 25 30Val Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn
Thr His Leu Leu 35 40 45Leu Leu Lys Asp Ala Gln Val Phe Gly Glu Glu
Trp Gly Tyr Ser Ser 50 55 60Glu Asp Val Ala Glu Phe Tyr His Arg Gln
Leu Lys Leu Thr Gln Gln65 70 75 80Tyr Thr Asp His Cys Val Asn Trp
Tyr Asn Val Gly Leu Asn Gly Leu 85 90 95Arg Gly Ser Thr Tyr Asp Ala
Trp Val Lys Phe Asn Arg Phe Arg Arg 100 105 110Glu Met Thr Leu Thr
Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr 115 120 125Asp Ile Arg
Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp 130 135 140Ile
Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly145 150
155 160Pro Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu
Phe 165 170 175Asp Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln
Pro Gly Tyr 180 185 190Phe Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly
Asn Tyr Val Glu Thr 195 200 205Arg Pro Ser Ile Gly Ser Ser Lys Thr
Ile Thr Ser Pro Phe Tyr Gly 210 215 220Asp Lys Ser Thr Glu Pro Val
Gln Lys Leu Ser Phe Asp Gly Gln Lys225 230 235 240Val Tyr Arg Thr
Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly 245 250 255Lys Val
Tyr Leu Gly
Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp 260 265 270Gln Lys Asn
Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn 275 280 285Gly
His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr 290 295
300Thr Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr
Ala305 310 315 320Glu Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile
Pro Phe Phe Thr 325 330 335Trp Thr His Arg Ser Val Asp Phe Phe Asn
Thr Ile Asp Ala Glu Lys 340 345 350Ile Thr Gln Leu Pro Val Val Lys
Ala Tyr Ala Leu Ser Ser Gly Ala 355 360 365Ser Ile Ile Glu Gly Pro
Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu 370 375 380Lys Glu Ser Ser
Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser385 390 395 400Ala
Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr 405 410
415Thr Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val
420 425 430Ile Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr
Tyr Gln 435 440 445Thr Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly
Phe Ser Gly Asp 450 455 460Lys Asn Glu Leu Ile Ile Gly Ala Glu Ser
Phe Val Ser Asn Glu Lys465 470 475 480Ile Tyr Ile Asp Lys Ile Glu
Phe Ile Pro Val Gln Leu 485 4907123DNAArtificial sequenceSynthetic
Oligonucleotide 71agacaactct acagtaaaag atg 237220DNAArtificial
sequenceSynthetic Oligonucleotide 72ggtaattggt caatagaatc
207339DNAArtificial sequenceSynthetic Oligonucleotide 73cagaagatgt
tgctgaattc nnncatagac aattaaaac 397434DNAArtificial
sequenceSynthetic Oligonucleotide 74gatgttgctg aattctatnn
nagacaatta aaac 347533DNAArtificial sequenceSynthetic
Oligonucleotide 75cccattttat gatattnnnt tatactcaaa agg
337664DNAArtificial sequenceSynthetic Oligonucleotide 76agctatgctg
gtctcggaag aaannnnnnn nnnnnnnnnn nnnnaaaaga agccaagatc 60gaat
647740DNAArtificial sequenceSynthetic Oligonucleotide 77ggtcacctag
gtctctcttc caggaattta acgcattaac 407865DNAArtificial
sequenceSynthetic Oligonucleotide 78agctatgctg gtctcccatt
tnnnnnnnnn nnnnnnnnnn nnnnnnnngt taaaacagaa 60ctaac
657936DNAArtificial sequenceSynthetic Oligonucleotide 79atccagtggg
gtctcaaatg ggaaaagtac aattag 368063DNAArtificial sequenceSynthetic
Oligonucleotide 80catttttacg gatccaattt ttnnnnnnnn nnnnnnnnnn
nnnnnnggac caactttttt 60gag 638162DNAArtificial sequenceSynthetic
Oligonucleotide 81gaatttcata cgcgtcttca acctggtnnn nnnnnnnnnn
nntctttcaa ttattggtct 60gg 628273DNAArtificial sequenceSynthetic
Oligonucleotide 82aaaagtttat cgaactatag ctaatacaga cgtagcggct
nnnnnnnnnn nnnnngtata 60tttaggtgtt acg 738320DNAArtificial
sequenceSynthetic Oligonucleotide 83ggagttccat ttgctggggc
208417DNAArtificial sequenceSynthetic Oligonucleotide 84atctccataa
aatgggg 178532DNAArtificial sequenceSynthetic Oligonucleotide
85gcgaagtaaa agaagccaag gtcgaataag gg 328643DNAArtificial
sequenceSynthetic Oligonucleotide 86cctttaagtt tgcgaaatcc
acacagccaa ggtcgaataa ggg 438735DNAArtificial sequenceSynthetic
Oligonucleotide 87cccattttat gatgttcggt tatacccaaa agggg
358825DNAArtificial sequenceSynthetic Oligonucleotide 88ggccaagtga
agacccatgg aaggc 258922DNAArtificial sequenceSynthetic
Oligonucleotide 89gcagtttccg gattcgaagt gc 229017DNAArtificial
sequenceSynthetic Oligonucleotide 90ccgctacgtc tgtatta
179117DNAArtificial sequenceSynthetic Oligonucleotide 91ataatggaag
cacctga 179260DNAArtificial sequenceSynthetic Oligonucleotide
92agctatgctg gtctcttctt annnnnnnnn nnnnnnnnna caattccatt ttttacttgg
609340DNAArtificial sequenceSynthetic Oligonucleotide 93atccagttgg
gtctctaaga aacaaaccgc gtaattaagc 409420DNAArtificial
sequenceSynthetic Oligonucleotide 94cctcaagggt tataacatcc
209555DNAArtificial sequenceSynthetic Oligonucleotide 95gtacaaaagc
taagctttnn nnnnnnnnnn nnnnnncgaa ctatagctaa tacag 55967PRTBacillus
thuringiensis 96Ser Lys Arg Ser Gln Asp Arg1 5971959DNABacillus
thuringiensisCDS(1)..(1956) 97atg aat cca aac aat cga agt gaa cat
gat acg ata aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His
Asp Thr Ile Lys Val Thr Pro1 5 10 15aac agt gaa ttg caa act aac cat
aat caa tat cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His
Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa
tta aat tat aaa gaa ttt tta aga atg 144Pro Asn Ser Thr Leu Glu Glu
Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45act gaa gac agt tct acg
gaa gtg cta gac aac tct aca gta aaa gat 192Thr Glu Asp Ser Ser Thr
Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60gca gtt ggg aca gga
att tct gtt gta ggg cag att tta ggt gtt gta 240Ala Val Gly Thr Gly
Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80gga gtt cca
ttt gct ggg gca ctc act tca ttt tat caa tca ttt ctt 288Gly Val Pro
Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95aac act
ata tgg cca agt gat gct gac cca tgg aag gct ttt atg gca 336Asn Thr
Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105
110caa gtt gaa gta ctg ata gat aag aaa ata gag gag tat gct aaa agt
384Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser
115 120 125aaa gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa
gat tat 432Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu
Asp Tyr 130 135 140gtt aat gcg tta aat tcc tgg aag aaa aca cct tta
agt ttg cga agt 480Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu
Ser Leu Arg Ser145 150 155 160aaa aga agc caa gat cga ata agg gaa
ctt ttt tct caa gca gaa agt 528Lys Arg Ser Gln Asp Arg Ile Arg Glu
Leu Phe Ser Gln Ala Glu Ser 165 170 175cat ttt cgt aat tcc atg ccg
tca ttt gca gtt tcc aaa ttc gaa gtg 576His Phe Arg Asn Ser Met Pro
Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190ctg ttt cta cca aca
tat gca caa gct gca aat aca cat tta ttg cta 624Leu Phe Leu Pro Thr
Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205tta aaa gat
gct caa gtt ttt gga gaa gaa tgg gga tat tct tca gaa 672Leu Lys Asp
Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220gat
gtt gct gaa ttt tat cat aga caa tta aaa ctt aca caa caa tac 720Asp
Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230
235 240act gac cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta
aga 768Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu
Arg 245 250 255ggt tca act tat gat gca tgg gtc aaa ttt aac cgt ttt
cgc aga gaa 816Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe
Arg Arg Glu 260 265 270atg act tta act gta tta gat cta att gta ctt
ttc cca ttt tat gat 864Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu
Phe Pro Phe Tyr Asp 275 280 285att cgg tta tac tca aaa ggg gtt aaa
aca gaa cta aca aga gac att 912Ile Arg Leu Tyr Ser Lys Gly Val Lys
Thr Glu Leu Thr Arg Asp Ile 290 295 300ttt acg gat cca att ttt tca
ctt aat act ctt cag gag tat gga cca 960Phe Thr Asp Pro Ile Phe Ser
Leu Asn Thr Leu Gln Glu Tyr Gly Pro305 310 315 320act ttt ttg agt
ata gaa aac tct att cga aaa cct cat tta ttt gat 1008Thr Phe Leu Ser
Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335tat tta
cag ggg att gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056Tyr Leu
Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345
350ggg aaa gat tct ttc aat tat tgg tct ggt aat tat gta gaa act aga
1104Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg
355 360 365cct agt ata gga tct agt aag aca att act tcc cca ttt tat
gga gat 1152Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr
Gly Asp 370 375 380aaa tct act gaa cct gta caa aag cta agc ttt gat
gga caa aaa gtt 1200Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp
Gly Gln Lys Val385 390 395 400tat cga act ata gct aat aca gac gta
gcg gct tgg ccg aat ggt aag 1248Tyr Arg Thr Ile Ala Asn Thr Asp Val
Ala Ala Trp Pro Asn Gly Lys 405 410 415gta tat tta ggt gtt acg aaa
gtt gat ttt agt caa tat gat gat caa 1296Val Tyr Leu Gly Val Thr Lys
Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430aaa aat gaa act agt
aca caa aca tat gat tca aaa aga aac aat ggc 1344Lys Asn Glu Thr Ser
Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445cat gta agt
gca cag gat tct att gac caa tta ccg cca gaa aca aca 1392His Val Ser
Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460gat
gaa cca ctt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440Asp
Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470
475 480tgt ttc tta atg cag gac cgt cgt gga aca att cca ttt ttt act
tgg 1488Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr
Trp 485 490 495aca cat aga agt gta gac ttt ttt aat aca att gat gct
gaa aag att 1536Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala
Glu Lys Ile 500 505 510act caa ctt cca gta gtg aaa gca tat gcc ttg
tct tca ggt gct tcc 1584Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu
Ser Ser Gly Ala Ser 515 520 525att att gaa ggt cca gga ttc aca gga
gga aat tta cta ttc cta aaa 1632Ile Ile Glu Gly Pro Gly Phe Thr Gly
Gly Asn Leu Leu Phe Leu Lys 530 535 540gaa tct agt aat tca att gct
aaa ttt aaa gtt aca tta aat tca gca 1680Glu Ser Ser Asn Ser Ile Ala
Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560gcc ttg tta caa
cga tat cgt gta aga ata cgc tat gct tct acc act 1728Ala Leu Leu Gln
Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575aac tta
cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtc atc 1776Asn Leu
Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585
590tac att aat aaa act atg aat aaa gat gat gat tta aca tat caa aca
1824Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr
595 600 605ttt gat ctc gca act act aat tct aat atg ggg ttc tcg ggt
gat aag 1872Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly
Asp Lys 610 615 620aat gaa ctt ata ata gga gca gaa tct ttc gtt tct
aat gaa aaa atc 1920Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser
Asn Glu Lys Ile625 630 635 640tat ata gat aag ata gaa ttt atc cca
gta caa ttg taa 1959Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu
645 65098652PRTBacillus thuringiensis 98Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg Ser Gln Asp Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile Arg Leu Tyr Ser Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro
Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro305 310 315 320Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe
340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His
Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455
460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile
580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Lys 610 615 620Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630
635 640Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645
650992000DNAArtificial sequenceRecombinant delta endotoxin
99ccatccatgg caaaccctaa caatcgttcc gaacacgaca ccatcaaggt tactccaaac
60tctgagttgc aaactaatca caaccagtac ccattggctg acaatcctaa cagtactctt
120gaggaactta actacaagga gtttctccgg atgaccgaag atagctccac
tgaggttctc 180gataactcta cagtgaagga cgctgttgga actggcatta
gcgttgtggg acagattctt 240ggagtggttg gtgttccatt cgctggagct
ttgaccagct tctaccagtc ctttctcaac 300accatctggc cttcagatgc
tgatccctgg aaggctttca tggcccaagt ggaagtcttg 360atcgataaga
agatcgaaga gtatgccaag tctaaagcct tggctgagtt gcaaggtttg
420cagaacaact tcgaggatta cgtcaacgca ctcaacagct ggaagaaaac
tcccttgagt 480ctcaggtcta agcgttccca ggaccgtatt cgtgaacttt
tcagccaagc cgaatcccac 540ttcagaaact ccatgcctag ctttgccgtt
tctaagttcg aggtgctctt cttgccaaca 600tacgcacaag ctgccaacac
tcatctcttg cttctcaaag acgctcaggt gtttggtgag 660gaatggggtt
actccagtga agatgttgcc gagttctacc gtaggcagct caagttgact
720caacagtaca cagaccactg cgtcaactgg tacaacgttg ggctcaatgg
tcttagagga 780tctacctacg acgcatgggt gaagttcaac aggtttcgta
gagagatgac cttgactgtg 840ctcgatctta tcgttctctt tccattctac
gacattcgtc tttactccaa aggcgttaag 900acagagctga ccagagacat
cttcaccgat cccatcttcc tacttacgac cctgcagaaa 960tacggtccaa
cttttctctc cattgagaac agcatcagga agcctcacct cttcgactat
1020ctgcaaggca ttgagtttca caccaggttg caacctggtt acttcggtaa
ggattccttc 1080aactactgga gcggaaacta cgttgaaacc agaccatcca
tcggatctag caagaccatc 1140acttctccat tctacggtga caagagcact
gagccagtgc agaagttgag cttcgatggg 1200cagaaggtgt atagaaccat
cgccaatacc gatgttgcag cttggcctaa tggcaaggtc 1260taccttggag
ttactaaagt ggacttctcc caatacgacg atcagaagaa cgagacatct
1320actcaaacct acgatagtaa gaggaacaat ggccatgttt ccgcacaaga
ctccattgac 1380caacttccac ctgaaaccac tgatgaacca ttggagaagg
cttacagtca ccaacttaac 1440tacgccgaat gctttctcat gcaagacagg
cgtggcacca ttccgttctt tacatggact 1500cacaggtctg tcgacttctt
taacactatc gacgctgaga agattaccca acttcccgtg 1560gtcaaggctt
atgccttgtc cagcggagct tccatcattg aaggtccagg cttcaccggt
1620ggcaacttgc tcttccttaa ggagtccagc aactccatcg ccaagttcaa
agtgacactt 1680aactcagcag ccttgctcca acgttacagg gttcgtatca
gatacgcaag cactaccaat 1740cttcgcctct ttgtccagaa cagcaacaat
gatttccttg tcatctacat caacaagact 1800atgaacaaag acgatgacct
cacctaccaa acattcgatc ttgccactac caatagtaac 1860atgggattct
ctggtgacaa gaacgagctg atcataggtg ctgagagctt tgtctctaat
1920gagaagattt acatagacaa gatcgagttc attccagttc aactctaata
gatcccccgg 1980gctgcaggaa ttcgatatca 2000100653PRTArtificial
sequenceRecombinant delta endotoxin 100Met Ala Asn Pro Asn Asn Arg
Ser Glu His Asp Thr Ile Lys Val Thr1 5 10 15Pro Asn Ser Glu Leu Gln
Thr Asn His Asn Gln Tyr Pro Leu Ala Asp 20 25 30Asn Pro Asn Ser Thr
Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg 35 40 45Met Thr Glu Asp
Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys 50 55 60Asp Ala Val
Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val65 70 75 80Val
Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe 85 90
95Leu Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met
100 105 110Ala Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr
Ala Lys 115 120 125Ser Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn
Asn Phe Glu Asp 130 135 140Tyr Val Asn Ala Leu Asn Ser Trp Lys Lys
Thr Pro Leu Ser Leu Arg145 150 155 160Ser Lys Arg Ser Gln Asp Arg
Ile Arg Glu Leu Phe Ser Gln Ala Glu 165 170 175Ser His Phe Arg Asn
Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu 180 185 190Val Leu Phe
Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu 195 200 205Leu
Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser 210 215
220Glu Asp Val Ala Glu Phe Tyr Arg Arg Gln Leu Lys Leu Thr Gln
Gln225 230 235 240Tyr Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly
Leu Asn Gly Leu 245 250 255Arg Gly Ser Thr Tyr Asp Ala Trp Val Lys
Phe Asn Arg Phe Arg Arg 260 265 270Glu Met Thr Leu Thr Val Leu Asp
Leu Ile Val Leu Phe Pro Phe Tyr 275 280 285Asp Ile Arg Leu Tyr Ser
Lys Gly Val Lys Thr Glu Leu Thr Arg Asp 290 295 300Ile Phe Thr Asp
Pro Ile Phe Leu Leu Thr Thr Leu Gln Lys Tyr Gly305 310 315 320Pro
Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe 325 330
335Asp Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr
340 345 350Phe Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val
Glu Thr 355 360 365Arg Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser
Pro Phe Tyr Gly 370 375 380Asp Lys Ser Thr Glu Pro Val Gln Lys Leu
Ser Phe Asp Gly Gln Lys385 390 395 400Val Tyr Arg Thr Ile Ala Asn
Thr Asp Val Ala Ala Trp Pro Asn Gly 405 410 415Lys Val Tyr Leu Gly
Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp 420 425 430Gln Lys Asn
Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn 435 440 445Gly
His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr 450 455
460Thr Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr
Ala465 470 475 480Glu Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile
Pro Phe Phe Thr 485 490 495Trp Thr His Arg Ser Val Asp Phe Phe Asn
Thr Ile Asp Ala Glu Lys 500 505 510Ile Thr Gln Leu Pro Val Val Lys
Ala Tyr Ala Leu Ser Ser Gly Ala 515 520 525Ser Ile Ile Glu Gly Pro
Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu 530 535 540Lys Glu Ser Ser
Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser545 550 555 560Ala
Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr 565 570
575Thr Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val
580 585 590Ile Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr
Tyr Gln 595 600 605Thr Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly
Phe Ser Gly Asp 610 615 620Lys Asn Glu Leu Ile Ile Gly Ala Glu Ser
Phe Val Ser Asn Glu Lys625 630 635 640Ile Tyr Ile Asp Lys Ile Glu
Phe Ile Pro Val Gln Leu 645 6501012050DNABacillus thuringiensis
101tggagctcca ccgcggtggc ggccgctcta gaactagtgg atctaggcct
ccatatgaac 60cctaacaatc gttccgaaca cgacaccatc aaggttactc caaactctga
gttgcaaact 120aatcacaacc agtacccatt ggctgacaat cctaacagta
ctcttgagga acttaactac 180aaggagtttc tccggatgac cgaagatagc
tccactgagg ttctcgataa ctctacagtg 240aaggacgctg ttggaactgg
cattagcgtt gtgggacaga ttcttggagt ggttggtgtt 300ccattcgctg
gagctttgac cagcttctac cagtcctttc tcaacaccat ctggccttca
360gatgctgatc cctggaaggc tttcatggcc caagtggaag tcttgatcga
taagaagatc 420gaagagtatg ccaagtctaa agccttggct gagttgcaag
gtttgcagaa caacttcgag 480gattacgtca acgcactcaa cagctggaag
aaaactccct tgagtctcag gtctaagcgt 540tcccaggacc gtattcgtga
acttttcagc caagccgaat cccacttcag aaactccatg 600cctagctttg
ccgtttctaa gttcgaggtg ctcttcttgc caacatacgc acaagctgcc
660aacactcatc tcttgcttct caaagacgct caggtgtttg gtgaggaatg
gggttactcc 720agtgaagatg ttgccgagtt ctaccatagg cagctcaagt
tgactcaaca gtacacagac 780cactgcgtca actggtacaa cgttgggctc
aatggtctta gaggatctac ctacgacgca 840tgggtgaagt tcaacaggtt
tcgtagagag atgaccttga ctgtgctcga tcttatcgtt 900ctctttccat
tctacgacat tcgtctttac tccaaaggcg ttaagacaga gctgaccaga
960gacatcttca ccgatcccat cttctcactt aacaccctgc aggaatacgg
tccaactttt 1020ctctccattg agaacagcat caggaagcct cacctcttcg
actatctgca aggcattgag 1080tttcacacca ggttgcaacc tggttacttc
ggtaaggatt ccttcaacta ctggagcgga 1140aactacgttg aaaccagacc
atccatcgga tctagcaaga ccatcacttc tccattctac 1200ggtgacaaga
gcactgagcc agtgcagaag ttgagcttcg atgggcagaa ggtgtataga
1260accatcgcca ataccgatgt tgcagcttgg cctaatggca aggtctacct
tggagttact 1320aaagtggact tctcccaata cgacgatcag aagaacgaga
catctactca aacctacgat 1380agtaagagga acaatggcca tgtttccgca
caagactcca ttgaccaact tccacctgaa 1440accactgatg aaccattgga
gaaggcttac agtcaccaac ttaactacgc cgaatgcttt 1500ctcatgcaag
acaggcgtgg caccattccg ttctttacat ggactcacag gtctgtcgac
1560ttctttaaca ctatcgacgc tgagaagatt acccaacttc ccgtggtcaa
ggcttatgcc 1620ttgtccagcg gagcttccat cattgaaggt ccaggcttca
ccggtggcaa cttgctcttc 1680cttaaggagt ccagcaactc catcgccaag
ttcaaagtga cacttaactc agcagccttg 1740ctccaacgtt acagggttcg
tatcagatac gcaagcacta ccaatcttcg cctctttgtc 1800cagaacagca
acaatgattt ccttgtcatc tacatcaaca agactatgaa caaagacgat
1860gacctcacct acaacacatt cgatcttgcc actaccaata gtaacatggg
attctctggt 1920gacaagaacg agctgatcat aggtgctgag agctttgtct
ctaatgagaa gatttacata 1980gacaagatcg agttcattcc agttcaactc
taatagatcc cccgggctgc aggaattcga 2040tatcaagctt
20501022280DNABacillus thuringiensis 102ttaaaattaa ttttgtatac
ttttcattgt aataatatga ttttaaaaac gaaaaagtgc 60atatacaact tatcaggagg
ggggggatgc acaaagaaga aaagaataag aagtgaatgt 120ttataatgtt
caatagtttt atgggaaggc attttatcag gtagaaagtt atgtattatg
180ataagaatgg gaggaagaaa aatgaatcca aacaatcgaa gtgaacatga
tacgataaag 240gttacaccta acagtgaatt gcaaactaac cataatcaat
atcctttagc tgacaatcca 300aattcaacac tagaagaatt aaattataaa
gaatttttaa gaatgactga agacagttct 360acggaagtgc tagacaactc
tacagtaaaa gatgcagttg ggacaggaat ttctgttgta 420gggcagattt
taggtgttgt aggagttcca tttgctgggg cactcacttc attttatcaa
480tcatttctta acactatatg gccaagtgat gctgacccat ggaaggcttt
tatggcacaa 540gttgaagtac tgatagataa gaaaatagag gagtatgcta
aaagtaaagc tcttgcagag 600ttacagggtc ttcaaaataa tttcgaagat
tatgttaatg cgttaaattc ctggaagaaa 660acacctttaa gtttgcgaag
taaaagaagc caagatcgaa taagggaact tttttctcaa 720gcagaaagtc
attttcgtaa ttccatgccg tcatttgcag tttccaaatt cgaagtgctg
780tttctaccaa catatgcaca agctgcaaat acacatttat tgctattaaa
agatgctcaa 840gtttttggag aagaatgggg atattcttca gaagatgttg
ctgaatttta tcatagacaa 900ttaaaactta cacaacaata cactgaccat
tgtgttaatt ggtataatgt tggattaaat 960ggtttaagag gttcaactta
tgatgcatgg gtcaaattta accgttttcg cagagaaatg 1020actttaactg
tattagatct aattgtactt ttcccatttt atgatattcg gttatactca
1080aaaggggtta aaacagaact aacaagagac atttttacgg atccaatttt
ttcacttaat 1140actcttcagg agtatggacc aacttttttg agtatagaaa
actctattcg aaaacctcat 1200ttatttgatt atttacaggg gattgaattt
catacgcgtc ttcaacctgg ttactttggg 1260aaagattctt tcaattattg
gtctggtaat tatgtagaaa ctagacctag tataggatct 1320agtaagacaa
ttacttcccc attttatgga gataaatcta ctgaacctgt acaaaagcta
1380agctttgatg gacaaaaagt ttatcgaact atagctaata cagacgtagc
ggcttggccg 1440aatggtaagg tatatttagg tgttacgaaa gttgatttta
gtcaatatga tgatcaaaaa 1500aatgaaacta gtacacaaac atatgattca
aaaagaaaca atggccatgt aagtgcacag 1560gattctattg accaattacc
gccagaaaca acagatgaac cacttgaaaa agcatatagt 1620catcagctta
attacgcgga atgtttctta atgcaggacc gtcgtggaac aattccattt
1680tttacttgga cacatagaag tgtagacttt tttaatacaa ttgatgctga
aaagattact 1740caacttccag tagtgaaagc atatgccttg tcttcaggtg
cttccattat tgaaggtcca 1800ggattcacag gaggaaattt actattccta
aaagaatcta gtaattcaat tgctaaattt 1860aaagttacat taaattcagc
agccttgtta caacgatatc gtgtaagaat acgctatgct 1920tctaccacta
acttacgact ttttgtgcaa aattcaaaca atgattttct tgtcatctac
1980attaataaaa ctatgaataa agatgatgat ttaacatatc aaacatttga
tctcgcaact 2040actaattcta atatggggtt ctcgggtgat aagaatgaac
ttataatagg agcagaatct 2100ttcgtttcta atgaaaaaat ctatatagat
aagatagaat ttatcccagt acaattgtaa 2160ggagatttta aaatgttggg
tgatggtcaa aatgaaagaa taggaaggtg aattttgatg 2220gttaggaaag
attcttttaa caaaagcaac atggaaaagt atacagtaca aatattaacc
228010332DNAArtificial sequenceSynthetic Oligonucleotide
103taggcctcca tccatggcaa accctaacaa tc 3210442DNAArtificial
sequenceSynthetic Oligonucleotide 104tcccatcttc ctacttacga
ccctgcagaa atacggtcca ac 4210528DNAArtificial sequenceSynthetic
Oligonucleotide 105gacctcacct accaaacatt cgatcttg
2810625DNAArtificial sequenceSynthetic Oligonucleotide
106cgagttctac cgtaggcagc tcaag 251071959DNAArtificial
sequenceRecombinant delta endotoxin 107atgaatccaa acaatcgaag
tgaacatgat acgataaagg ttacacctaa cagtgaattg 60caaactaacc ataatcaata
tcctttagct gacaatccaa attcaacact agaagaatta 120aattataaag
aatttttaag aatgactgaa gacagttcta cggaagtgct agacaactct
180acagtaaaag atgcagttgg gacaggaatt tctgttgtag ggcagatttt
aggtgttgta 240ggagttccat ttgctggggc actcacttca ttttatcaat
catttcttaa cactatatgg 300ccaagtgatg ctgacccatg gaaggctttt
atggcacaag ttgaagtact gatagataag 360aaaatagagg agtatgctaa
aagtaaagct cttgcagagt tacagggtct tcaaaataat 420ttcgaagatt
atgttaatgc gttaaattcc tggaagaaaa cacctttaag tttgcgaagt
480aaaagaagcc aaggtcgaat aagggaactt ttttctcaag cagaaagtca
ttttcgtaat 540tccatgccgt catttgcagt ttccaaattc gaagtgctgt
ttctaccaac atatgcacaa 600gctgcaaata cacatttatt gctattaaaa
gatgctcaag tttttggaga agaatgggga 660tattcttcag aagatgttgc
tgaattctat cgtagacaat taaaacttac acaacaatac 720actgaccatt
gtgttaattg gtataatgtt ggattaaatg gtttaagagg ttcaacttat
780gatgcatggg tcaaatttaa ccgttttcgc agagaaatga ctttaactgt
attagatcta 840attgtacttt tcccatttta tgatattcgg ttatactcaa
aaggggttaa aacagaacta 900acaagagaca tttttacgga tccaattttt
ttacttacta cgcttcagaa gtacggacca 960acttttttga gtatagaaaa
ctctattcga aaacctcatt tatttgatta tttacagggg 1020attgaatttc
atacgcgtct tcaacctggt tactttggga aagattcttt caattattgg
1080tctggtaatt atgtagaaac tagacctagt ataggatcta gtaagacaat
tacttcccca 1140ttttatggag ataaatctac tgaacctgta caaaagctaa
gctttgatgg acaaaaagtt 1200tatcgaacta tagctaatac agacgtagcg
gcttggccga atggtaaggt atatttaggt 1260gttacgaaag ttgattttag
tcaatatgat gatcaaaaaa atgaaactag tacacaaaca 1320tatgattcaa
aaagaaacaa tggccatgta agtgcacagg attctattga ccaattaccg
1380ccagaaacaa cagatgaacc acttgaaaaa gcatatagtc atcagcttaa
ttacgcggaa 1440tgtttcttaa tgcaggaccg tcgtggaaca attccatttt
ttacttggac acatagaagt 1500gtagactttt ttaatacaat tgatgctgaa
aagattactc aacttccagt agtgaaagca 1560tatgccttgt cttcaggtgc
ttccattatt gaaggtccag gattcacagg aggaaattta 1620ctattcctaa
aagaatctag taattcaatt gctaaattta aagttacatt aaattcagca
1680gccttgttac aacgatatcg tgtaagaata cgctatgctt ctaccactaa
cttacgactt 1740tttgtgcaaa attcaaacaa tgattttctt gtcatctaca
ttaataaaac tatgaataaa 1800gatgatgatt taacatatca aacatttgat
ctcgcaacta ctaattctaa tatggggttc 1860tcgggtgata agaatgaact
tataatagga gcagaatctt tcgtttctaa tgaaaaaatc 1920tatatagata
agatagaatt tatcccagta caattgtaa 1959108652PRTArtificial
sequenceRecombinant delta endotoxin 108Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg Ser Gln Gly Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220Asp Val Ala Glu Phe Tyr Arg Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile Arg Leu Tyr Ser Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro
Ile Phe Leu Leu Thr Thr
Leu Gln Lys Tyr Gly Pro305 310 315 320Thr Phe Leu Ser Ile Glu Asn
Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335Tyr Leu Gln Gly Ile
Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp
Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365Pro
Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375
380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys
Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp
Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val Thr Lys Val Asp Phe
Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu Thr Ser Thr Gln Thr
Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His Val Ser Ala Gln Asp
Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460Asp Glu Pro Leu
Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480Cys
Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490
495Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile
500 505 510Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly
Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu
Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys
Val Thr Leu Asn Ser Ala545 550 555 560Ala Leu Leu Gln Arg Tyr Arg
Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575Asn Leu Arg Leu Phe
Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn
Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605Phe
Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615
620Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys
Ile625 630 635 640Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu
645 650109649PRTArtificial sequenceRecombinant delta endotoxin
109Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Ala Thr Glu1
5 10 15Asn Asn Glu Val Ser Asn Asn His Ala Gln Tyr Pro Leu Ala Asp
Thr 20 25 30Pro Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Arg
Thr Thr 35 40 45Asp Asn Asn Val Glu Ala Leu Asp Ser Ser Thr Thr Lys
Asp Ala Ile 50 55 60Gln Lys Gly Ile Ser Ile Ile Gly Asp Leu Leu Gly
Val Val Gly Phe65 70 75 80Pro Tyr Gly Gly Ala Leu Val Ser Phe Tyr
Thr Asn Leu Leu Asn Thr 85 90 95Ile Trp Pro Gly Glu Asp Pro Leu Lys
Ala Phe Met Gln Gln Val Glu 100 105 110Ala Leu Ile Asp Gln Lys Ile
Ala Asp Tyr Ala Lys Asp Lys Ala Thr 115 120 125Ala Glu Leu Gln Gly
Leu Lys Asn Val Phe Lys Asp Tyr Val Ser Ala 130 135 140Leu Asp Ser
Trp Asp Lys Thr Pro Leu Thr Leu Arg Asp Gly Arg Ser145 150 155
160Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His Phe Arg
165 170 175Arg Ser Met Pro Ser Phe Ala Val Ser Gly Tyr Glu Val Leu
Phe Leu 180 185 190Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu
Leu Leu Lys Asp 195 200 205Ala Gln Ile Tyr Gly Thr Asp Trp Gly Tyr
Ser Thr Asp Asp Leu Asn 210 215 220Glu Phe His Thr Lys Gln Lys Asp
Leu Thr Ile Glu Tyr Thr Asn His225 230 235 240Cys Ala Lys Trp Tyr
Lys Ala Gly Leu Asp Lys Leu Arg Gly Ser Thr 245 250 255Tyr Glu Glu
Trp Val Lys Phe Asn Arg Tyr Arg Arg Glu Met Thr Leu 260 265 270Thr
Val Leu Asp Leu Ile Thr Leu Phe Pro Leu Tyr Asp Val Arg Thr 275 280
285Tyr Thr Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Val Leu Thr Asp
290 295 300Pro Ile Val Ala Val Asn Asn Met Asn Gly Tyr Gly Thr Thr
Phe Ser305 310 315 320Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu
Phe Asp Tyr Leu His 325 330 335Ala Ile Gln Phe His Ser Arg Leu Gln
Pro Gly Tyr Phe Gly Thr Asp 340 345 350Ser Phe Asn Tyr Trp Ser Gly
Asn Tyr Val Ser Thr Arg Ser Ser Ile 355 360 365Gly Ser Asp Glu Ile
Ile Arg Ser Pro Phe Tyr Gly Asn Lys Ser Thr 370 375 380Leu Asp Val
Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Phe Arg Ala385 390 395
400Val Ala Asn Gly Asn Leu Ala Val Trp Pro Val Gly Thr Gly Gly Thr
405 410 415Lys Ile His Ser Gly Val Thr Lys Val Gln Phe Ser Gln Tyr
Asn Asp 420 425 430Arg Lys Asp Glu Val Arg Thr Gln Thr Tyr Asp Ser
Lys Arg Asn Val 435 440 445Gly Gly Ile Val Phe Asp Ser Ile Asp Gln
Leu Pro Pro Ile Thr Thr 450 455 460Asp Glu Ser Leu Glu Lys Ala Tyr
Ser His Gln Leu Asn Tyr Val Arg465 470 475 480Cys Phe Leu Leu Gln
Gly Gly Arg Gly Ile Ile Pro Val Phe Thr Trp 485 490 495Thr His Lys
Ser Val Asp Phe Tyr Asn Thr Leu Asp Ser Glu Lys Ile 500 505 510Thr
Gln Ile Pro Phe Val Lys Ala Phe Ile Leu Val Asn Ser Thr Ser 515 520
525Val Val Ala Gly Pro Gly Phe Thr Gly Gly Asp Ile Ile Lys Cys Thr
530 535 540Asn Gly Ser Gly Leu Thr Leu Tyr Val Thr Pro Ala Pro Asp
Leu Thr545 550 555 560Tyr Ser Lys Thr Tyr Lys Ile Arg Ile Arg Tyr
Ala Ser Thr Ser Gln 565 570 575Val Arg Phe Gly Ile Asp Leu Gly Ser
Tyr Thr His Ser Ile Ser Tyr 580 585 590Phe Asp Lys Thr Met Asp Lys
Gly Asn Thr Leu Thr Tyr Asn Ser Phe 595 600 605Asn Leu Ser Ser Val
Ser Arg Pro Ile Glu Ile Ser Gly Gly Asn Lys 610 615 620Ile Gly Val
Ser Val Gly Gly Ile Gly Ser Gly Asp Glu Val Tyr Ile625 630 635
640Asp Lys Ile Glu Phe Ile Pro Met Asp 645110652PRTArtificial
sequenceRecombinant delta endotoxin 110Met Asn Pro Asn Asn Arg Ser
Glu His Asp Thr Ile Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Pro Thr
Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu
Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser
Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly
Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly
Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90
95Asp Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala
100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala
Lys Ser 115 120 125Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn
Phe Glu Asp Tyr 130 135 140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr
Pro Leu Ser Leu Arg Ser145 150 155 160Lys Arg Ser Gln Asp Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser
Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu
Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215
220Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln
Tyr225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu
Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe
Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu
Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Val Arg Leu Tyr Ser Lys
Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro
Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro305 310 315 320Thr
Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330
335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Ser
340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu
Thr Arg 355 360 365Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro
Phe Tyr Gly Asp 370 375 380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
Phe Asp Gly Gln Lys Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr
Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415Ile Tyr Phe Gly Val
Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu
Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His
Val Gly Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455
460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala
Glu465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro
Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe Phe Asn Thr
Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys Ala
Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly
Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn
Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560Ala
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570
575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Ile Val Ile
580 585 590Tyr Ile Asn Lys Thr Met Asn Ile Asp Asp Asp Leu Thr Tyr
Gln Thr 595 600 605Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe
Ser Gly Asp Thr 610 615 620Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
Val Ser Asn Glu Lys Ile625 630 635 640Tyr Ile Asp Lys Ile Glu Phe
Ile Pro Val Gln Leu 645 650111652PRTArtificial sequenceRecombinant
delta endotoxin 111Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile
Lys Val Thr Pro1 5 10 15Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr
Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr
Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu
Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val
Val Gly Gln Ile Leu Gly Val Val65 70 75 80Gly Val Pro Phe Ala Gly
Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95Asn Thr Ile Trp Pro
Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110Gln Val Glu
Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125Lys
Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135
140Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg
Ser145 150 155 160Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser
Gln Ala Glu Ser 165 170 175His Phe Arg Asn Ser Met Pro Ser Phe Ala
Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln
Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu Lys Asp Ala Gln Val
Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220Asp Val Ala Glu
Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240Thr
Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250
255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu
260 265 270Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe
Tyr Asp 275 280 285Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu
Thr Arg Asp Ile 290 295 300Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr
Leu Gln Glu Tyr Gly Pro305 310 315 320Thr Phe Leu Ser Ile Glu Asn
Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335Tyr Leu Gln Gly Ile
Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp
Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365Pro
Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375
380Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys
Val385 390 395 400Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp
Pro Asn Gly Lys 405 410 415Val Tyr Leu Gly Val Thr Lys Val Asp Phe
Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu Thr Ser Thr Gln Thr
Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His Val Ser Ala Gln Asp
Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460Asp Glu Pro Leu
Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480Cys
Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490
495Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile
500 505 510Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly
Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu
Leu Phe Leu Lys 530 535 540Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys
Val Thr Leu Asn Ser Ala545 550 555 560Ala Leu Leu Gln Arg Tyr Arg
Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575Asn Leu Arg Leu Phe
Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn
Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605Phe
Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615
620Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys
Ile625 630 635 640Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu
645 650112659PRTArtificial sequenceRecombinant delta endotoxin
112Met Ile Arg Met Gly Gly Arg Lys Met Asn Pro Asn Asn Arg Ser Glu1
5 10 15Tyr Asp Thr Ile Lys Val Thr Pro Asn Ser Glu Leu Pro Thr Asn
His 20 25 30Asn Gln Tyr Pro Leu Ala Asp Asn Pro Asn Ser Thr Leu Glu
Glu Leu 35 40 45Asn Tyr Lys Glu Phe Leu Arg Met Thr Ala Asp Asn Ser
Thr Glu Val 50 55 60Leu Asp Ser Ser Thr Val Lys Asp Ala Val Gly Thr
Gly Ile Ser Val65 70 75 80Val Gly Gln Ile Leu Gly Val Val Gly Val
Pro Phe Ala Gly Ala Leu 85 90 95Thr Ser Phe Tyr Gln Ser Phe Leu Asn
Ala Ile Trp Pro Ser Asp Ala 100 105 110Asp Pro Trp Lys Ala Phe Met
Ala Gln Val Glu Val Leu Ile Asp Lys 115 120 125Lys Ile Glu Glu Tyr
Ala Lys Ser Lys Ala Leu Ala Glu Leu Gln Gly 130 135 140Leu Gln Asn
Asn Phe Glu Asp
Tyr Val Asn Ala Leu Asp Ser Trp Lys145 150 155 160Lys Ala Pro Val
Asn Leu Arg Ser Arg Arg Ser Gln Asp Arg Ile Arg 165 170 175Glu Leu
Phe Ser Gln Ala Glu Ser His Phe Arg Asn Ser Met Pro Ser 180 185
190Phe Ala Val Ser Lys Phe Glu Val Leu Phe Leu Pro Thr Tyr Ala Gln
195 200 205Ala Ala Asn Thr His Leu Leu Leu Leu Lys Asp Ala Gln Val
Phe Gly 210 215 220Glu Glu Trp Gly Tyr Ser Ser Glu Asp Ile Ala Glu
Phe Tyr Gln Arg225 230 235 240Gln Leu Lys Leu Thr Gln Gln Tyr Thr
Asp His Cys Val Asn Trp Tyr 245 250 255Asn Val Gly Leu Asn Ser Leu
Arg Gly Ser Thr Tyr Asp Ala Trp Val 260 265 270Lys Phe Asn Arg Phe
Arg Arg Glu Met Thr Leu Thr Val Leu Asp Leu 275 280 285Ile Val Leu
Phe Pro Phe Tyr Asp Val Arg Leu Tyr Ser Lys Gly Val 290 295 300Lys
Thr Glu Leu Thr Arg Asp Ile Phe Thr Asp Pro Ile Phe Thr Leu305 310
315 320Asn Ala Leu Gln Glu Tyr Gly Pro Thr Phe Ser Ser Ile Glu Asn
Ser 325 330 335Ile Arg Lys Pro His Leu Phe Asp Tyr Leu Arg Gly Ile
Glu Phe His 340 345 350Thr Arg Leu Arg Pro Gly Tyr Ser Gly Lys Asp
Ser Phe Asn Tyr Trp 355 360 365Ser Gly Asn Tyr Val Glu Thr Arg Pro
Ser Ile Gly Ser Asn Asp Thr 370 375 380Ile Thr Ser Pro Phe Tyr Gly
Asp Lys Ser Ile Glu Pro Ile Gln Lys385 390 395 400Leu Ser Phe Asp
Gly Gln Lys Val Tyr Arg Thr Ile Ala Asn Thr Asp 405 410 415Ile Ala
Ala Phe Pro Asp Gly Lys Ile Tyr Phe Gly Val Thr Lys Val 420 425
430Asp Phe Ser Gln Tyr Asp Asp Gln Lys Asn Glu Thr Ser Thr Gln Thr
435 440 445Tyr Asp Ser Lys Arg Tyr Asn Gly Tyr Leu Gly Ala Gln Asp
Ser Ile 450 455 460Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu
Glu Lys Ala Tyr465 470 475 480Ser His Gln Leu Asn Tyr Ala Glu Cys
Phe Leu Met Gln Asp Arg Arg 485 490 495Gly Thr Ile Pro Phe Phe Thr
Trp Thr His Arg Ser Val Asp Phe Phe 500 505 510Asn Thr Ile Asp Ala
Glu Lys Ile Thr Gln Leu Pro Val Val Lys Ala 515 520 525Tyr Ala Leu
Ser Ser Gly Ala Ser Ile Ile Glu Gly Pro Gly Phe Thr 530 535 540Gly
Gly Asn Leu Leu Phe Leu Lys Glu Ser Ser Asn Ser Ile Ala Lys545 550
555 560Phe Lys Val Thr Leu Asn Ser Ala Ala Leu Leu Gln Arg Tyr Arg
Val 565 570 575Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Leu Phe
Val Gln Asn 580 585 590Ser Asn Asn Asp Phe Leu Val Ile Tyr Ile Asn
Lys Thr Met Asn Ile 595 600 605Asp Gly Asp Leu Thr Tyr Gln Thr Phe
Asp Phe Ala Thr Ser Asn Ser 610 615 620Asn Met Gly Phe Ser Gly Asp
Thr Asn Asp Phe Ile Ile Gly Ala Glu625 630 635 640Ser Phe Val Ser
Asn Glu Lys Ile Tyr Ile Asp Lys Ile Glu Phe Ile 645 650 655Pro Val
Gln113652PRTArtificial sequenceRecombinant delta endotoxin 113Met
Ile Arg Lys Gly Gly Arg Lys Met Asn Pro Asn Asn Arg Ser Glu1 5 10
15His Asp Thr Ile Lys Thr Thr Glu Asn Asn Glu Val Pro Thr Asn His
20 25 30Val Gln Tyr Pro Leu Ala Glu Thr Pro Asn Pro Thr Leu Glu Asp
Leu 35 40 45Asn Tyr Lys Glu Phe Leu Arg Met Thr Ala Asp Asn Asn Thr
Glu Ala 50 55 60Leu Asp Ser Ser Thr Thr Lys Asp Val Ile Gln Lys Gly
Ile Ser Val65 70 75 80Val Gly Asp Leu Leu Gly Val Val Gly Phe Pro
Phe Gly Gly Ala Leu 85 90 95Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr
Ile Trp Pro Ser Glu Asp 100 105 110Pro Trp Lys Ala Phe Met Glu Gln
Val Glu Ala Leu Met Asp Gln Lys 115 120 125Ile Ala Asp Tyr Ala Lys
Asn Lys Ala Leu Ala Glu Leu Gln Gly Leu 130 135 140Gln Asn Asn Val
Glu Asp Tyr Val Ser Ala Leu Ser Ser Trp Gln Lys145 150 155 160Asn
Pro Val Ser Ser Arg Asn Pro His Ser Gln Gly Arg Ile Arg Glu 165 170
175Leu Phe Ser Gln Ala Glu Ser His Phe Arg Asn Ser Met Pro Ser Phe
180 185 190Ala Ile Ser Gly Tyr Glu Val Leu Phe Leu Thr Thr Tyr Ala
Gln Ala 195 200 205Ala Asn Thr His Leu Phe Leu Leu Lys Asp Ala Gln
Ile Tyr Gly Glu 210 215 220Glu Trp Gly Tyr Glu Lys Glu Asp Ile Ala
Glu Phe Tyr Lys Arg Gln225 230 235 240Leu Lys Leu Thr Gln Glu Tyr
Thr Asp His Cys Val Lys Trp Tyr Asn 245 250 255Val Gly Leu Asp Lys
Leu Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn 260 265 270Phe Asn Arg
Tyr Arg Arg Glu Met Thr Leu Thr Val Leu Asp Leu Ile 275 280 285Ala
Leu Phe Pro Leu Tyr Asp Val Arg Leu Tyr Pro Lys Glu Val Lys 290 295
300Thr Glu Leu Thr Arg Asp Val Leu Thr Asp Pro Ile Val Gly Val
Asn305 310 315 320Asn Leu Arg Gly Tyr Gly Thr Thr Phe Ser Asn Ile
Glu Asn Tyr Ile 325 330 335Arg Lys Pro His Leu Phe Asp Tyr Leu His
Arg Ile Gln Phe His Thr 340 345 350Arg Phe Gln Pro Gly Tyr Tyr Gly
Asn Asp Ser Phe Asn Tyr Trp Ser 355 360 365Gly Asn Tyr Val Ser Thr
Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile 370 375 380Thr Ser Pro Phe
Tyr Gly Asn Lys Ser Ser Glu Pro Val Gln Asn Leu385 390 395 400Glu
Phe Asn Gly Glu Lys Val Tyr Arg Ala Val Ala Asn Thr Asn Leu 405 410
415Ala Val Trp Pro Ser Ala Val Tyr Ser Gly Val Thr Lys Val Glu Phe
420 425 430Ser Gln Tyr Asn Asp Gln Thr Asp Glu Ala Ser Thr Gln Thr
Tyr Asp 435 440 445Ser Lys Arg Asn Val Gly Ala Val Ser Trp Asp Ser
Ile Asp Gln Leu 450 455 460Pro Pro Glu Thr Thr Asp Glu Pro Leu Glu
Lys Gly Tyr Ser His Gln465 470 475 480Leu Asn Tyr Val Met Cys Phe
Leu Met Gln Gly Ser Arg Gly Thr Ile 485 490 495Pro Val Leu Thr Trp
Thr His Lys Ser Val Asp Phe Phe Asn Met Ile 500 505 510Asp Ser Lys
Lys Ile Thr Gln Leu Pro Leu Val Lys Ala Tyr Lys Leu 515 520 525Gln
Ser Gly Ala Ser Val Val Ala Gly Pro Arg Phe Thr Gly Gly Asp 530 535
540Ile Ile Gln Cys Thr Glu Asn Gly Ser Ala Ala Thr Ile Tyr Val
Thr545 550 555 560Pro Asp Val Ser Tyr Ser Gln Lys Tyr Arg Ala Arg
Ile His Tyr Ala 565 570 575Ser Thr Ser Gln Ile Thr Phe Thr Leu Ser
Leu Asp Gly Ala Pro Phe 580 585 590Asn Gln Tyr Tyr Phe Asp Lys Thr
Ile Asn Lys Gly Asp Thr Leu Thr 595 600 605Tyr Asn Ser Phe Asn Leu
Ala Ser Phe Ser Thr Pro Phe Glu Leu Ser 610 615 620Gly Asn Asn Leu
Gln Ile Gly Val Thr Gly Leu Ser Ala Gly Asp Lys625 630 635 640Val
Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Asn 645 650
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References