U.S. patent application number 15/141179 was filed with the patent office on 2016-08-11 for peptide toxin formulation.
The applicant listed for this patent is Vestaron Corporation. Invention is credited to Peter Carlson, John McIntyre, Daniel Russell, William Tedford.
Application Number | 20160227766 15/141179 |
Document ID | / |
Family ID | 42058098 |
Filed Date | 2016-08-11 |
United States Patent
Application |
20160227766 |
Kind Code |
A1 |
Tedford; William ; et
al. |
August 11, 2016 |
PEPTIDE TOXIN FORMULATION
Abstract
Procedures are described which use solvents to increase the
topical insecticidal activity of toxic insect peptides. These
procedures comprise drying the peptides, if needed, followed by the
addition of either: 1) a polar organic solvent, with or without
water, to a dried peptide, or 2) the addition of polar aprotic
solvent or other adjuvant to the dried peptide, followed by the
addition of either: 1) a polar organic solvent, with or without
water, (where a polar aprotic solvent is added first) or 2) a polar
aprotic solvent or other adjuvant to the peptide polar organic
solvent (where the polar organic solvent is added first), to the
peptide formulation.
Inventors: |
Tedford; William; (Calgary,
CA) ; McIntyre; John; (Alto, MI) ; Russell;
Daniel; (St. Louis, MO) ; Carlson; Peter;
(Potomac, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vestaron Corporation |
Kalamazoo |
MI |
US |
|
|
Family ID: |
42058098 |
Appl. No.: |
15/141179 |
Filed: |
April 28, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14200893 |
Mar 7, 2014 |
9352022 |
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15141179 |
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13768984 |
Feb 15, 2013 |
8703910 |
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14200893 |
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13528402 |
Jun 20, 2012 |
8501684 |
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13768984 |
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12568400 |
Sep 28, 2009 |
8217003 |
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13528402 |
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61101825 |
Oct 1, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01N 37/18 20130101;
A01N 37/46 20130101; A01N 63/10 20200101; A01N 25/02 20130101; A61K
38/1767 20130101; C07K 14/43522 20130101; C07K 14/43518 20130101;
A01N 37/18 20130101; A01N 25/02 20130101; A01N 37/46 20130101; A01N
25/02 20130101; A01N 63/10 20200101; A01N 25/02 20130101; A01N
37/18 20130101; A01N 2300/00 20130101; A01N 63/10 20200101; A01N
25/02 20130101 |
International
Class: |
A01N 25/02 20060101
A01N025/02; A01N 63/02 20060101 A01N063/02; A01N 37/18 20060101
A01N037/18 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 28, 2009 |
US |
PCT/US2009/058603 |
Claims
1. A process for increasing the topical insecticidal activity of a
peptide that is toxic to insects comprising making a topical toxic
peptide formulation comprised of the following three components
mixed together; 1) a peptide that is toxic to insects with 2) a
polar aprotic solvent and 3) a polar organic solvent according to
the following procedure: a) selecting a peptide that is toxic to
insects and that is less than about 200 amino acids and greater
than about 10 amino acids; and b) mixing a first type of solvent
with said peptide, wherein said first type of solvent is either a
polar aprotic solvent or a polar organic solvent, to make a first
solvent peptide mixture; c) mixing a second type of solvent with
said first solvent peptide mixture, wherein said second type of
solvent is not the same type of solvent as said first type of
solvent and is either a polar aprotic solvent or a polar organic
solvent, to make a topical toxic peptide formulation; wherein said
polar aprotic solvent is selected from: dimethyl sulfoxide (DMSO).,
dimethylformamide, dioxane and hexamethylphosphorotriamide and
wherein said polar organic solvent is selected from propanol and
all its isomers, methyl ethyl ketone, diethyl ketone, acetonitrile,
and ethyl acetoacetate saving said topical toxic peptide
formulation.
2. (canceled)
3. (canceled)
4. The process of claim 1 wherein said polar aprotic solvent is
selected from: dimethyl sulfoxide (DMSO) or MSO.RTM. and said polar
organic solvent is propanol.
5. The process of claim 1 wherein said peptide is lyophylized
before the first solvent is mixed with the peptide.
6. The process of claim 1 wherein said first solvent is a polar
aprotic solvent.
7. The process of claim 1 wherein said polar aprotic solvent is
from about 60% to about 99% percent, and said polar organic solvent
is about 40% to about 1%, of the total solvent volume.
8. The process of claim 1 wherein said polar aprotic solvent is
from about 75% to about 95%, and the polar organic solvent is about
25 to about 5%, of the total solvent volume.
9. The process of claim 1 wherein said polar aprotic solvent is
from about 80% to about 90% of the total solvent volume and the
polar organic solvent is from about 20% to about 10% of the total
solvent volume.
10. The process of claim 1 wherein said peptide is any topical
toxic peptide selected from a selected or derived from a toxic
peptide from any species of spider, scorpion, snake, mite, snail,
slug or plant and any peptides having 50% or greater homology to
any such peptide.
11. The process of claim 1 wherein the length of said peptide is
from about 20 to less than about 100 amino acids in length.
12. The process of claim 1 wherein said peptide has from 1-5,
internal difulfide bonds.
13. The process of claim 1 wherein said peptide is selected from a
spider or scorpion.
14. The process of claim 1 wherein said peptide is selected from
the Australian funnel web spider of genus Atrax or Hadronyche.
15. The process of claim 1 wherein said peptide is selected from
any sequence in the sequence listing or any sequence having 50% or
greater homology to any of the listed sequences.
16. The process of claim 15 wherein said peptide is selected from
any of the sequences in the sequence listing.
17. The process of claim 16 wherein said peptide is selected from
any of the following sequences: SEQ ID NO: 60, SEQ ID NO: 117, SEQ
ID NO: 118, or SEQ ID NO: 119.
18. A topical toxic peptide formulation made according to the
process of claim 1 and comprising the following: a) a peptide toxic
to insects, as defined in claim 1; b) a polar organic solvent, as
defined in claim 1; c) a polar aprotic solvent or adjuvant, as
defined in claim 1; d) wherein said polar organic solvent comprises
from about 70 to about 99 percent (%) of the final volume of the
formulation; e) wherein said polar aprotic solvent or adjuvant
comprises from about 30, to about 1 percent (%) of the final volume
of the formulation; f) an optional water phase, wherein said water
phase comprises from 0 (zero), to about 10 percent (%) of the final
volume of the formulation.
19. A topical toxic peptide formulation as described in, claim 18,
wherein said topical toxic peptide is from about 20 to less than
about 100 amino acids in length.
20. The control of an insect with the topical toxic peptide
formulation of claim 18 wherein the topical toxic peptide
formulation is applied to the insect's environment.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 13/528,402, filed Jun. 20, 2012, which is a
divisional of U.S. application Ser. No. 12/568,400, filed Sep. 28,
2009, issued as U.S. Pat. No. 8,271,003, which claims the priority
of U.S. Provisional Application No. 61/101,825, filed Oct. 1, 2008,
all of which are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] This invention relates to the field of formulations for
insecticidal peptides.
BACKGROUND
[0003] Insecticidal peptides are toxic to their targets when
delivered internally, but sometimes they have little or no topical
activity. Topical insecticidal activity refers to a toxin's ability
to inhibit the growth, impair the movement or even kill an insect
when the toxin is delivered to the insect or the insect's
environment by spraying, or other means, as opposed to delivering
the toxin directly to the insect's gut or internal organs by
injection or inducing the insect to consume the toxin from its
food, for example an insect feeding upon a transgenic plant.
[0004] The ability to successfully enhance or even change the
properties of peptides with solvents has, until now, proven
elusive. The wide variety, unique properties and special nature of
peptides, combined with the huge variety of possible solvents one
could choose from, has produced only a few described methods for
the enhancement of a few selected peptides in the past 50 years or
so. Various texts on the subject exist. See for example, Principles
of Dairy Chemistry Jenness and Patton (1959) pp. 115-117, 127, 317,
326-328, 333.
[0005] Attempts have been made to enhance the activity of a few
peptides through purification and extraction. For example, U.S.
Pat. No. 5,840,838, Hensley, describes a procedure for enhancing
the activity of amyloid .beta. peptide, a 39-43 residue peptide,
with a process that involves dissolving the peptide in an organic
solvent, incubating it for 45 minutes to 3 hours above room
temperature, equilibrating to room temperature and then removing
the solvent.
[0006] U.S. Pat. No. 4,530,784, Rosenberg, relates to a method of
extracting a biologically active factor that restores contact
inhibition of growth to malignant cells in mammals by mixing
specially prepared media with a volatile non-denaturing
precipitating agent. The precipitate formed by this reaction is
separated from the formulation and extracted with a biologically
acceptable ionic buffering agent.
[0007] U.S. Pat. No. 4,337,194, Diaz, is a process of preparing
somatostatin using a step-wise peptide coupling reaction in a
solution of DMF. The product of the reaction is isolated by
evaporation or by precipitation with a second solvent which renders
the somatostatin insoluble, then the crude peptide obtained is
purified.
[0008] There are few if any descriptions, however, for a method to
convert a peptide which has low topical insecticidal activity into
one having significantly greater topical insecticidal activity.
[0009] The procedure described here increases the topical
insecticidal toxicity of insecticidal peptides. Peptides thus
treated are referred to herein as "enhanced topical peptides." The
process described herein of making enhanced topical peptides is
sometimes called making the peptides "special." The process of
making the peptides special makes the peptides more active than
before they are treated with the process or treatment described
herein. Once the peptides have been made special they can be
applied topically to the insect, the insect's environment, to the
places it inhabits, its habitat and to the food it touches, eats or
consumes; in order to control the insect, rather than having to
engineer the peptide into the genome of a suitable plant or other
food. Both the new process, the formulations, and the new enhanced
topical peptides produced by the process are described and claimed
herein.
SUMMARY OF THE INVENTION
[0010] Procedures are described which use solvents to increase the
toxicity of toxic insect peptides. Those procedures involve the
preparation of the peptides by drying the peptides, if needed,
followed by the addition of either: 1) a polar organic solvent,
with or without water, to a dried peptide, or 2) a polar aprotic
solvent or other adjuvant to the dried peptide, followed by the
addition of either: 1) a polar organic solvent, with or without
water, (where a polar aprotic solvent is added first or 2) a polar
aprotic solvent or other adjuvant to the peptide polar organic
solvent (where the polar organic solvent is added first), to the
peptide formulation.
[0011] The procedures can also be described as follows: A method of
increasing the topical insecticidal activity of a toxic insect
peptide, herein called making the peptide special comprising:
adding either i) a polar organic solvent or ii) a polar aprotic
solvent or adjuvant to the peptide and then adding either i) a
polar organic solvent or ii) a polar aprotic solvent or adjuvant,
which ever was not added initially to the initial peptide
formulation of above.
[0012] A method is described herein where the polar organic solvent
comprises from about 50, to about 99.9 percent (%) of the final
volume of the formulation. The method is specifically described
where the polar organic solvent comprises from about 60, 70, 85, 90
to about 99.0 percent (%) of the final volume of the formulation.
The method is described wherein the polar organic solvent comprises
from about 70, to about 99.0 percent (%) of the final volume of the
formulation. The method is specifically described wherein the polar
organic solvent comprises from about 60, 70, 80, 85, 90, to about
99.0 percent (%) of the final volume of the formulation. The polar
organic solvent may be selected from acetone, methanol, ethanol,
propanol and all its isomers, methyl ethyl ketone, diethyl ketone,
acetonitrile, ethyl acetoacetate. The polar organic solvents
selected from acetone, methanol, ethanol, propanol and all its
isomers are especially useful.
[0013] The polar aprotic solvent or adjuvant will comprise from
about 20%, to about 0.001%, of the final volume of the formulation.
Specifically the polar aprotic solvent or adjuvant, comprises from
about 15%, to about 0.005%, from about 10%, to about 0.01%, from
about 8% , to about 0.1%, from about 5%, to about 0.1%, of the
final volume of the formulation. The polar aprotic solvent or
adjuvant is selected from dimethyl sulfoxide, dimethylformamide,
dioxane and hexamethylphosphorotriamide. Dimethyl sulfoxide, also
known as DMSO is exemplified.
[0014] The toxic insect peptides are preferably those with a)
greater than 10 amino acid residues and less than 3000 amino acid
residues; b) a molecular weight from about 550 Da to about 350,000
Da; and c) they have insecticidal activity. The peptides may
optionally have 1 to 5 disulfide bonds. The insecticidal activity
of the peptides optionally are peptides having topical activity in
at least one reproducable topical insecticidal assay. The toxic
insect peptides may be selected from the venom of a spider, mite,
scorpion, snake, snails, certain plants or any combination thereof.
The spider may be an Australian funnel web spider, and peptides
from the genus of Atrax or Hadronyche are easily made special using
the procedures described herein. Specific peptides from spiders,
scorpions and plants are provided in the sequence listing.
[0015] Disclosed are formulations of special toxic peptides
comprising: a) a peptide; b) a polar organic solvent; c) a polar
aprotic solvent or adjuvant; d) wherein said polar organic solvent
comprises from about 80, to about 99 percent (%) of the final
volume of the formulation; e) wherein said polar aprotic solvent or
adjuvant comprises from about 1, to about 10 percent (%) of the
final volume of the suspension; and f) an optional water phase,
wherein said water phase comprises from 0 (zero), to about 10
percent (%) of the final volume of the suspension.
[0016] The peptides made special by the process of this invention
are new and may be separately claimed. These peptides are described
by all of their properties and not simply their sequence. For the
most part the peptide sequence information of the peptides which
can be made special, as described herein, are known; however, once
treated the same peptides will have greated topical activity. These
peptides made special are novel with unique properties, both the
peptides and the process of making them are disclosed and claimed
herein.
[0017] Methods to control insects are also disclosed, in particular
applications of the special toxic peptides or special toxic peptide
formulations applied to the insect's environment. The special toxic
peptide may be applied as a dry or liquid formulation. The
formulation may include wetting and dispersing agents, surfactants
and other common components of insecticidal peptide formulations.
Also, described are special toxic peptides produced as the product
of any of the processes described herein. The processes described
herein can be used with any peptides. The following peptides are
mentioned by way of specific examples and are not intended to limit
the range, type or number of peptides can be be made special using
this process.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0018] "Active ingredient" means a peptide or polypeptide, herein
it is sometimes called a toxin.
[0019] "Insecticidal activity", "insect control" or "control the
insect" means that on or after exposure of the insect to the active
ingredient, the insect either dies, stops or slows its movement or
its feeding, stops or slows its growth, fails to pupate, cannot
reproduce or cannot produce fertile offspring.
[0020] "Insect('s) environment" means any place or surface that is
or will be exposed to an insect. The insect's environment includes
the places it inhabits, its habitat and the food it touches, eats
or consumes.
[0021] "Toxic insect peptide" means a peptide having insecticidal
activity when ingested by or injected into an insect but having
little, low or no topical insecticidal activity.
[0022] "Peptide made special" means the same as "Special topical
peptide" below.
[0023] "Polar aprotic solvents" are organic solvents that have ion
dissolving power but lack an acidic hydrogen. A polar aprotic
solvent cannot donate hydrogen (H.sup.+ or proton.) These solvents
generally have high dielectric constants and high polarity. Further
examples that should be considered representative and not limiting
are; dimethyl sulfoxide (DMSO), dimethylformamide, dioxane,
hexamethylphosphorotriamide and methyl sulfoxide (MSO.RTM.).
Adjuvants as polar aprotic solvents. Certain adjuvants can also be
polar aprotic solvents. Mixtures of oils with surfactants, commonly
referred to as adjuvants are other examples of polar aprotic
solvents. Crop oils in combination with surfactants can also act as
polar aprotic solvents. Examples such as Agicide Activator.RTM.,
Herbimax.RTM., Maximizer.RTM., and MSO.RTM. all available from
Loveland company serve to demonstrate the commercial adjuvants can
act as the polar aprotic solvents as the term is defined by this
invention. MSO.RTM. is a methylated seed oil and surfactant blend
that uses methyl esters of soya oil in amounts of between about 80
and 85 percent petroleum oil with 15 to 20 percent surfactant. Use
and descriptions of MSO.RTM. used as a polar aprotic solvent can be
found in Example 7.
[0024] "Polar organic solvents" are organic solvents with dipole
moments sufficient to confer a dielectric constant of 15 or higher,
acetone being one example. Other examples of polar organic solvents
include compounds with a dissociable H.sup.+, such as lower alkyl
alcohols. Further examples that should be considered representative
and not limiting are as follows: acetone, methanol, ethanol,
propanol, all isomers of propanol including 1- and 2-, propanol (n-
and iso-propanol, respectively). Other polar organic solvents which
might be successfully used as part of this formulation can be
determined by those skilled in the art; these may include methyl
ethyl ketone, diethyl ketone, acetonitrile, ethyl acetoacetate,
etc.
[0025] "Special topical peptide" means a peptide previously having
low topical insecticidal activity that has relatively higher
topical insecticidal activity because of the procedures described
herein used to increase the topical activity of peptides.
[0026] "Topical activity" or "topical insecticidal activity" means
insecticidal activity that results from exposure or contact of the
insect's outer layers, to the insecticidal peptide. Topical
activity can result from exposure to or contact between a treated
material or surface and the external part of the insect, such as
its feet, abdomen, antenna, mouth. Topical activity can result from
insect preening of external parts of the insect followed by
ingestion of the toxin. Treatment of any material or surface with
insecticide or toxin that then comes in contact with the insect
with resultant insecticidal activity is considered a topical
activity.
[0027] "Topical application" means the application of the active
ingredient to an insect's environment, or the insect itself. An
insect's environment may be treated with a "topical application" in
any manner including: spraying, painting, baiting, impregnating
materials, such as treating paper or other objects that are then
placed in the same area when the insect is known or expected to
visit or frequent. Topical application can also mean direct contact
with the insect and the insecticide.
The Process to Make Special
[0028] Description of the process to make peptides special.
[0029] Add a polar organic solvent, with or without water, to a
dried peptide and then add a polar aprotic solvent or other
adjuvant to the peptide polar organic solvent (optional water)
formulation, or in the alternative first add a polar aprotic
solvent or other adjuvant to a dried peptide and then add a polar
organic solvent (with or without water) to the polar aprotic
solvent peptide formulation. Additional treatments and
pretreatments to the peptides and peptide solvent formulations are
optional and are discussed below.
[0030] The peptides made special are then used as desired for
effect. Application and use of the peptides made special may be
with any means, either standard or as determined to be effective by
a practicioner who is skilled in the art, including but not limited
to: spraying through an atomizer or other type of spray nozzle,
direct/indirect application of droplets of the formulation,
application of the dried residue of the formulation to any body
surface of the targeted insect, immersion of the targeted insect in
a bath, etc.
[0031] The peptide is made special upon completion of the addition
of the polar organic solvent and the polar aprotic solvent or other
adjuvant, with the solvents added in either order. It is better to
start with a peptide that is in a non-aqueous environment. The
preferred order of solvent addition will be determined by a
practicioner who is skilled in the art of insecticidal formulation,
giving attention and consideration to the particular peptides used.
Numerous variations of the manner in which the solvent is added can
be made and should be apparent to one skilled in the art. Some
variations and more details of the procedure are provided
below.
[0032] Preparation of the peptide by removing water may be needed
if the starting peptides are dissolved in water. The procedure of
making the peptides special may be practiced with peptides having
either high or low solubility in water. Often peptides are prepared
in water based solvents or expressed in aqueous environments. If
the peptide to be made special is in an aqueous environment, most
of the water should first be removed, i.e. the peptide should be
dried. If the peptide is already in a dried state, then drying is
not needed. Preparation of peptides can involve concentrating,
purifying, isolating or identifying peptides and or the amounts or
concentration of the peptide in the sample. Once the peptide is in
a preferred state, condition or concentration, it should be
"dried." One method of drying a peptide, or taking it out of an
aqueous environment, is to lyophilize the peptide using traditional
peptide lyophilization procedures. See Protein Analysis and
Purification 2.sup.nd Ed. Rosenberg 2005 pp 140. Other methods
include, but are not limited to, spray drying, rotary evaporation,
and vacuum centrifugation. Peptide drying should be done in a
manner such that the peptides are not unduly damaged or destroyed.
Excessive heat should be avoided. Those skilled in the art will
know and be able to practice appropriate procedures to dry the
peptide.
[0033] Adding the polar organic solvent to the dried peptide. Once
the peptide is prepared by having most of the water removed, it is
ready for mixing with either the polar organic solvent or the polar
aprotic solvent or adjuvant. Better results are sometimes obtained
when the polar organic solvent is added to the dried peptide before
the polar aprotic solvent is added and order is described below,
but with some peptides in some situations the polar aprotic solvent
is added to the dried peptide followed by addition of the polar
organic solvent.
The Polar Organic Solvent.
[0034] Many polar organic solvents can be used, some seem to
produce peptides having greater topical activity than others. We
have found the following polar organic solvents work well in this
procedure to make peptides special: acetone, methanol, ethanol,
propanol, all isomers of propanol including 1- and 2-, propanol (n-
and iso-propanol, respectively). Other polar organic solvents which
might be successfully used as part of this formulation can be
determined by those skilled in the art; these may include methyl
ethyl ketone, diethyl ketone, acetonitrile, ethyl acetoacetate,
etc. The mixing of peptide and solvent can be done with any
laboratory method of mixing such as vortex mixing, stirring,
shaking, etc.
[0035] If the polar organic solvent is added to the dried peptide
before the aprotic solvent/adjuvant then it (the polar organic
solvent) can be as much as about 98 to 100% of the liquid in the
formulation, and the peptide will likely form a precipitate in the
solvent. The formulation may appear as a cloudy or hazy suspension.
It should be vigorously mixed. The polar organic solvent can have
some water in it, see "Water" below, but pure or dry solvent also
works well. Optimal final concentrations of water and polar organic
solvent can be determined for a formulation of a particular special
topical peptide by those skilled in the art. We have formulated
special topical peptides with polar organic solvent at final
concentrations of 80 to 99%. Lower concentrations than 80% would
also work with some peptides. We specifically describe polar
organic solvents at final concentrations of 60, 70, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 and 99%
and with water at final concentrations of 0% to 10%, in particular
final water concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% are
described. Those skilled in the art will be able to successfully
use values outside these sample ranges for particular formulations
of particular special topical peptides.
Sonication.
[0036] Once the peptide is properly taken up in the solvent it may
be sonicated or otherwise treated to further increase its topical
activity. Sonication may break up the peptide particles in solution
and reduce the size of the suspended particles. Sonication or other
procedures to reduce particle size appears to increase topical
toxicity of the peptides. Other procedures that reduce particle
size, in addition to sonication, may be used to increase topical
activity. Various procedures to reduce particle size should be
known to those familiar with peptide manipulation. Without being
limited to any particular procedure or mechanism, using a high
speed blender, shaking or stirring with glass beads may also be
useful to increase the toxicity of the special toxic peptides.
Water.
[0037] As mentioned above, the polar organic solvent does not need
to be pure or absolute when used: it can contain water. Moreover,
this water may contain salts, organic molecules, peptides, etc.
Water can presumably be added to the peptide either before the
polar organic solvent is added to the peptide formulation, or at
the same time or after addition of the polar organic solvent. Care
should be taken not to use too large a concentration of water, as
we have observed that this can reduce or eliminate the activity of
certain formulations of certain special topical peptides. The water
need not be pure, it can include various proteins such as
chitinases, phospholipases, lectins, etc., or salts, sugars,
carbohydrates, etc., in order to create a more useful and stable
final solution. Alternatively, the water phase can initially be
pure water and then various proteins such as chitinases,
phospholipases, lectins etc. could be added to the water phase
after it is mixed with the polar organic solvent and the
peptides.
The Polar Aprotic Solvent.
[0038] The polar aprotic solvent or adjuvant can be used as the
first ingredient added to the dried peptide or it can be added to
the peptide polar organic solvent (water optional) formulation
described above. Those skilled in the art can determine whether the
polar aprotic solvent or the polar organic solvent should be the
first liquid added to the dried peptide in order to determine the
better way to make the peptides special. This determination will
depend on the circumstances of each case and in particular exactly
which toxic insect peptide is used and the final formulation
desired. However, such variations should be practiced with care, as
we have observed that in some cases lower insecticidal activity
resulted if the polar aprotic solvent was added to the peptide
before the polar organic solvent is added.
[0039] Polar aprotic solvents lack an acidic hydrogen. These
solvents generally have high dielectric constants and high
polarity. Examples include dimethyl sulfoxide, dimethylformamide,
dioxane and hexamethylphosphorotriamide.
[0040] The adjuvant can be any oil and/or emulsifying surfactant
formulated for agricultural application of pesticides and
especially peptides. These commercial formulations typically have
oils and emulsifying surfactants formulated to "carry and spread"
the active ingredients. Examples include: "Aero Dyneamic" from
Helena Chemical Co. which has methylated or ethylated vegetable
oil, a nonionic surfactant and a buffering agent or acidifier. It
is further described as a "proprietary blend of ethoxylated alkyl
phosphate esters, polyalkylene modified polydimethylsiloxane,
nonionic emulsifiers and methylated vegetable oils. For aerial use
only at 2-8 qt/100 gal. 30-70 percent. Provides pH reduction and
buffering, NIS and oil blend" See label for rates. Further examples
and manufactures of adjuvants can be found in Table 1.
TABLE-US-00001 TABLE 1 Agrochemical Surfactants.
Surfactant/Adjuvant Composition Brief Description Suggested
Application LI 700 Phosphatidylcholine, Non-ionic low 8-24 oz/100
gallon (Loveland methylacetic acid, and foaming penetrant;
(0.0625%-0.1825% Products) alkyl polyoxyethylene aids in providing
solution) ether (80%); Constituents uniform spray ineffective as
spray coverage and to adjuvant (20%) acidify spray solutions SILWET
L-77 Polysiloxane polyether Non-ionic, 3-16 oz/100 gal (Loveland
copolymer, polyether organofunctional (0.02%-0.125% Products)
(100%) silicone surfactant solution) which lower's surface tension
below commonly used surfactants, resulting in more effective
wetting and more uniform coverage MSO Concentrate Methylated
vegetable oil, Enhances activity of 1-2 pints per acre w/LECI-TECH
alcohol ethoxylate, post applied (1.25-2.5% based on (Loveland
phosphatidylcholine herbicides non-ionic 10 gal/acre) Products)
(100%) surfactants and petroleum-based crop oils TACTIC Synthetic
latex, 1,2- Increases adherence 8-32 oz/100 gal. (Loveland
propanediol, Alcohol (latex polymer) and (0.0625%-0.25% Products)
ethoxylate, silicone coverage solution) polyether copolymer
(organosilicone) (63.4%); Constituents ineffective as spray
adjuvant (36.6%)
[0041] Optimal final concentrations of polar aprotic solvent and/or
adjuvant can be determined for the formulation of a particular
topical peptide made special by those skilled in the art. We have
successfully formulated special topical peptides with polar aprotic
solvent at final concentrations of 10% and as low as 0.01%, with
0.5% working well. The adjuvant Silwet L-77, for example, works
well at a final concentration as low as 0.01%, and those skilled in
the art should be able to successfully find other adjuvants using
even higher or lower values than the ranges described here for
particular formulations of particular topical peptides made
special.
[0042] The water and sonication steps described above can be
applied in any order. Particular modes of insecticidal application
for particular formulations of special topical peptides will be
determined by those skilled in the art.
Topical Toxic Peptides and their Preparation.
[0043] Examples of toxic insect peptides are well known and can be
found in numerous references. They can be identified by their
peptidic nature and their activity, usually oral or injection
insecticidal activity. Here we provide a few examples to better
illustrate and describe the invention, but the invention is not
limited to these examples. All of these examples and others not
shown here are descriptive of new materials, described and claimed
here for the first time.
[0044] Toxic insect peptides are peptides of greater than 5 amino
acid residues and less than 3000 amino acid residues. They range in
molecular weight from about 550 Da to about 350,000 Da. Toxic
insect peptides have some type of insecticidal activity. Typically
they show activity when injected into insects but most do not have
significant activity when applied to an insect topically. The
insecticidal activity of toxic insect peptides is measured in a
variety of ways. Common methods of measurement are widely known to
those skilled in the art. Such methods include, but are not limited
to determination of median response doses (e.g., LD.sub.50,
PD.sub.50, LC.sub.50, ED.sub.50) by fitting of dose-response plots
based on scoring various parameters such as: paralysis, mortality,
failure to gain weight, etc. Measurements can be made for cohorts
of insects exposed to various doses of the insecticidal formulation
in question. Analysis of the data can be made by creating curves
defined by probit analysis and/or the Hill Equation, etc. In such
cases, doses would be administered by hypodermic injection, by
hyperbaric infusion, by presentation of the insecticidal
formulation as part of a sample of food or bait, etc.
[0045] Toxic insect peptides are defined here as all peptides shown
to be insecticidal upon delivery to insects either by hypodermic
injection, hyperbaric infusion, or upon per os delivery to an
insect (i.e., by ingestion as part of a sample of food presented to
the insect). This class of peptides thus comprises, but is not
limited to, many peptides produced naturally as components of the
venoms of spiders, mites, scorpions, snakes, snails, etc. This
class also comprises, but is not limited to, various peptides
produced by plants (e.g., various lectins, ribosome inactivating
proteins, and cysteine proteases), and various peptides produced by
entomopathogenic microbes (e.g. the Cry1/delta endotoxin family of
proteins produced by various Bacillus species.)
[0046] The following documents are incorporated by reference in the
US in their entirely, in other jurisdictions where allowed and they
are of common knowledge given their publication. In addition they
are incorporated by reference and known specifically for their
sequence listings to the extent they describe peptide sequences.
See the following:
US Patents: U.S. Pat. No. 5,763,568, issued Jun. 9, 1998,
specifically the sequences in the sequence listing, and those
numbered 1-26, and those known as "kappa" or "omega" toxins,
including those that can form 2-4 intrachain disulphide bridges,
and the peptides appearing on columns 2 and 4, and Table 5, and in
FIG. 5, FIG. 15, FIG. 16, FIG. 17, FIG. 18. U.S. Pat. No.
5,959,182, issued Sep. 28, 1999, specifically the sequences in the
sequence listing, and those numbered 1-26 and those known as
"kappa" or "omega" toxins, including toxins that can form 2-4
intrachain disulphide bridges, and the peptides appearing on
columns 2 and 4, and Table 5, and in FIG. 5, FIG. 15, FIG. 16, FIG.
17, FIG. 18. U.S. Pat. No. 6,583,264 B2, issued Jun. 24, 2003, and
U.S. Pat. No. 7,173,106 B2, issued Feb. 6, 2007 specifically
sequence number 1, named "omega-atracotoxin-Hv2a or
.omega.-atracotoxin-Hv2a, including toxins that can form 2-4
intrachain disulphide bridges. U.S. Pat. No. 7,279,547 B2, issued
Oct. 9, 2007, specifically the sequences in the sequence listing,
and those numbered 1-35, and variants of .omega.-atracotoxin-Hv2a,
toxins that can form 2-4 intrachain disulphide bridges, and the
peptides appearing on columns 4-8 of the specification, and in FIG.
3 and FIG. 4. U.S. Pat. No. 7,354,993 B2, issued Apr. 8, 2008
specifically the peptide sequences listed in the sequence listing,
and those numbered 1-39, and those named U-ACTX polypeptides,
toxins that can form 2-4 intrachain disulphide bridges, and
variants thereof, and the peptides appearing on columns 4-9 of the
specification and in FIG. 1. EP patent 1 812 464 B1, published and
granted Aug. 10, 2008 Bulletin 2008/41, specifically the peptide
sequences listed in the sequence listing, toxins that can form 2-4
intrachain disulphide bridges, and those as numbered 1-39, and
those named U-ACTX polypeptides, and variants thereof, and the
peptides appearing in paragraphs 0023 to 0055, and appearing in
FIG. 1.
[0047] Described and incorporated by reference to the peptides
identified herein are homologous variants of sequences mentioned,
have homology to such sequences or referred to herein which are
also identified and claimed as suitable for making special
according to the processes described herein including but not
limited to all homologous sequences including homologous sequences
having at least any of the following percent identities to any of
the sequences disclosed her or to any sequence incorporated by
reference: 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90% or 95% or greater identitiy to any and all sequences
identified in the patents noted above, and to any other sequence
identified herein, including each and every sequence in the
sequence listing of this application. When the term homologous or
homology is used herein with a number such as 30% or greater then
what is meant is percent identity or percent similarity between the
two peptides. When homologous or homology is used without a numeric
percent then it refers to two peptide sequences that are closely
related in the evolutionary or developmental aspect in that they
share common physical and functional aspects like topical toxicity
and similar size within 100% greater length or 50% shorter length
or peptide.
[0048] Described and incorporated by reference to the peptides
identified herein that are derived from any source mentioned in the
US and EP patent documents referred to above, including but not
limited to the following: Toxins isolated from plants and insects,
especially toxins from spiders, scorpions and plants that prey on
or defend themselves from insects, such as, funnel web spiders and
especially. Australian funnel web spiders, including toxins found
in, isolated from or derived from the genus Atrax or Hadronyche,
including the genus species, Hadronyche versuta, or the Blue
Mountain funnel web spider, Atrax robustus, Atrax formidabilis,
Atrax infensus including toxins known as "atracotoxins,"
"co-atracotoxins," "kappa" atracotoxins, "omega" atracotoxins also
known as co-atracotoxin, U-ACTX polypetides, U-ACTX-Hv1a,
rU-ACTX-Hv1a, rU-ACTX-Hv1b, or mutants or variants, especially
peptides of any of these types and especially those less than about
200 amino acids but greater than about 10 amino acids, and
especially peptides less than about 150 amino acids but greater
than about 20 amino acids, especially peptides less than about 100
amino acids but greater than about 25 amino acids, especially
peptides less than about 65 amino acids but greater than about 25
amino acids, especially peptides less than about 55 amino acids but
greater than about 25 amino acids, especially peptides of about 37
or 39 or about 36 to 42 amino acids, especially peptides with less
than about 55 amino acids but greater than about 25 amino acids,
especially peptides with less than about 45 amino acids but greater
than about 35 amino acids, especially peptides with less than about
115 amino acids but greater than about 75 amino acids, especially
peptides with less than about 105 amino acids but greater than
about 85 amino acids, especially peptides with less than about 100
amino acids but greater than about 90 amino acids, including
peptide toxins of any of the lengths mentioned here that can form
2, 3 and or 4 or more intrachain disulphide bridges, including
toxins that disrupt calcium channel currents, including toxins that
disrupt potassium channel currents, especially insect calcium
channels or hybrids thereof, especially toxins or variants thereof
of any of these types, and any combination of any of the types of
toxins described herein that have topical insecticidal activity,
can be made special by the processes described herein.
[0049] Venomous peptides from the Australian Funnel Web Spider,
genus Atrax and Hadronyche are particularly suitable and work well
when treated by the methods, procedures or processes described by
this invention. These spider peptides, like many other toxic
peptides, including especially are toxic scorpion and toxic plant
peptides, become topically active or toxic when treated by the
processes described by this invention. Examples of suitable
peptides tested and with data are provided herein. In addition to
the organisms mentioned above, the following species are also
specifically know to carry toxins suitable for being made special
by the process of this invention. The following species are
specifically named: Agelenopsis aperta, Androctonus australis
Hector, Antrax formidabillis, Antrax infensus, Atrax robustus,
Bacillus thuringiensis, Bothus martensii Karsch, Bothus occitanus
tunetanus, Buthacus arenicola, Buthotus judaicus, Buthus occitanus
mardochei, Centruroides noxius, Centruroides suffusus suffusus,
Hadronyche infensa, Hadronyche versuta, Hadronyche versutus,
Hololena curia, Hottentotta judaica, Leiurus quinquestriatus,
Leiurus quinquestriatus hebraeus, Leiurus quinquestriatus
quinquestriatus, Oldenlandia affinis, Scorpio maurus palmatus,
Tityus serrulatus, Tityus zulianu. Any peptidic toxins from any of
the genus listed above and or genus species are suitable for being
made special according to the process in this invention.
[0050] The Examples in this specification are not intended to, and
should not be used to limit the invention, they are provided only
to illustrate the invention.
[0051] As noted above, many peptides are suitable candidates as the
subject of the process to make special. The sequences noted above,
below and in the sequence listing are especially suitable peptides
that can be made special, and many of these have been made special
according to this invention with the results shown in the examples
below.
TABLE-US-00002 (one letter code) SEQ ID NO: 60 SPTCI PSGQP CPYNE
NCCSQ SCTFK ENENG NTVKR CD 1 5 10 15 20 25 30 35 37 (three letter
code) SEQ ID NO: 60 Ser Pro Thr Cys Ile Pro Ser Gly Gln Pro Cys 1 5
10 Pro Tyr Asn Glu Asn Cys Cys Ser Gln Ser Cys 15 20 Thr Phe Lys
Glu Asn Glu Asn Gly Asn Thr Val 25 30 Lys Arg Cys Asp 35 37 Named
".omega.-ACTX-Hv1a" it has disulfide bridges at positions: 4-18,
11-22 and 17-36. The molecular weight is 4096. (one letter code)
SEQ ID NO: 117 GSSPT CIPSG QPCPY NENCC SQSCT FKENE NGNTV KRCD 1 5
10 15 20 25 30 35 39 (three letter code) SEQ ID NO: 117 Gly Ser Ser
Pro Thr Cys Ile Pro Ser Gly Gln 1 5 10 Pro Cys Pro Tyr Asn Glu Asn
Cys Cys Ser Gln 15 20 Ser Cys Thr Phe Lys Glu Asn Glu Asn Gly Asn
25 30 Thr Val Lys Arg Cys Asp 35 39 Named ".omega.-ACTX-Hv1a+2" it
has disulfide bridges at positions: 6-20, 13-24 and 19-38. The
molecular weight is 4199. (one letter code) SEQ ID NO: 118 GSAIC
TGADR PCAAC CPCCP GTSCK AESNG VSYCR KDEP 1 5 10 15 20 25 30 35 39
(three letter code) SEQ ID NO: 118 Gly Ser Ala Ile Cys Thr Gly Ala
Asp Arg Pro 1 5 10 Cys Ala Ala Cys Cys Pro Cys Cys Pro Gly Thr 15
20 Ser Cys Lys Ala Glu Ser Asn Gly Val Ser Tyr 25 30 Cys Arg Lys
Asp Glu Pro 35 39 Named "r.kappa.-ACTX-Hv1c" it has disulfide
bridges at positions: 5-19, 12-24, 15-16, 18-34. The molecular
weight is 3912.15 (one letter code) SEQ ID NO: 119 GSQYC VPVDQ
PCSLN TQPCC DDATC TQERN ENGHT 1 5 10 15 20 25 30 35 VYYCR A 40 41
(three letter code) SEQ ID NO: 119 Gly Ser Gln Tyr Cys Val Pro Val
Asp Gln Pro 1 5 10 Cys Ser Leu Asn Thr Gln Pro Cys Cys Asp Asp 15
20 Ala Thr Cys Thr Gln Glu Arg Asn Glu Asn Gly 25 30 His Thr Val
Tyr Tyr Cys Arg Ala 35 40 41 Named "rU-ACTX-Hv1a ("Hybrid")+2" it
has disulfide bridges at positions: 5-20, 12-25, 19-39. The
molecular weight is 4570.51
Preparation of the Topical Toxic Peptides
[0052] The toxic peptides described above can be prepared in a
variety of ways and in some embodiments they need not be prepared
by any formal process. The peptides can simply be collected with or
without other impurities in a composition and utilized. In one
embodiment in which several Examples are provided below, the
peptides are lyophylized or they have some, most or nearly all
liquid removed prior to being made special. In some embodiments the
peptides are still wet and only excess liquid is removed. In some
embodiments the peptides are in aqueous solutions or in something
similar to an aqueous solution. The peptides need not be isolated
or purified prior to being made special.
Reproducable Assays to Measure Topical Insecticidal Activity.
[0053] The topical insecticidal activity of a peptide can be
measured and quantified. Numerous assays are available. Several
examples of reproducable assays useful to determine the topical
activity of a peptide are provided in the examples below. These
examples describe both peptide and assay in detail but they should
not be used to limit the scope of the claims or invention.
MATERIALS AND METHODS
Examples
Example 1
Topical Assay with Acetone and DMSO using House Fly
[0054] Toxin is .omega.-ACTX-Hv1a:
TABLE-US-00003 (SEQ ID NO: 60)
SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD
[0055] Synthetic. Molecular weight: 4050 Da. LD.sub.50 in
House-fly: 90.2 pmol/g
[0056] Administration and application of the formulation.
[0057] Insect is House fly (Musca domestica) from Benzon research
weighing between 12-20 mg (average mass 16 mg) would each receive 2
.mu.L micropipette applications of formulations onto the dorsal
thoracic surface of the body.
[0058] Toxin Doses:
[0059] .about.90,000 pmol/g .omega.-ACTX (1000x Injection LD50)
dissolved in 90% Acetone/10% DMSO or
[0060] DMSO (10%-20%) with 0.1% Tween 20,
[0061] .about.9,000 pmol/g (100.times. Injection LD50), and
[0062] .about.900 pmol/g (10.times. LD50) dissolved in DMSO
(10-20%) with 0.1% Tween 20.
Preparation of Water-Based Application Solutions
[0063] .omega.-ACTX/DMS0 stock--3.5 mg lyophilized .omega.-ACTX
(Auspep) massed and dissolved in 70 .mu.L DMSO (50 .mu.g/.mu.L
stock). Water+Tween stocks-1000 .mu.L aliquots of Tween 20 stocks
were prepared in water to the percent volume to volume values
(listed below) from a 1% Tween 20 stock (e.g. 111 .mu.L 1% Tween
20+889 .mu.L water for the 0.111% Tween 20 stock, etc.). In all
cases, the Tween 20 stock was added to the tube first, then DMSO,
and finally the .omega.-ACTX/DMSO stock.
TABLE-US-00004 TABLE 2 Water-DMSO Treatments. .omega.-ACTX Water +
Dose [DMSO] Tween Final (pmol/g) (%) .omega.-ACTX DMSO (% v/v)
Volume 90,000 10% 17.5 .mu.L .omega.-ACTX STOCK 12.5 .mu.L 270
.mu.L 300 .mu.L (50 .mu.g/.mu.L in DMSO) (0.111% Tween) -ve 10% --
30 .mu.L 270 .mu.L 300 .mu.L (0.111% Tween) 90,000 20% 17.5 .mu.L
.omega.-ACTX STOCK 42.5 .mu.L 240 .mu.L 300 .mu.L (50 .mu.g/.mu.L
in DMSO) (0.125% Tween) 9,000 20% 30 .mu.L 90,000 pmol/g ACTX 54
.mu.L 216 .mu.L 300 .mu.L Solution (0.138% Tween) 900 20% 30 .mu.L
9,000 pmol/g ACTX 54 .mu.L 216 .mu.L 300 .mu.L Solution (0.138%
Tween) -ve 20% -- 60 .mu.L 240 .mu.L 300 .mu.L (0.125% Tween) Note.
In Table 2 and many Tables below some or all of the following
abbreviations are used: "twitch" or "twch" means twitching; "morb"
means moribund and "-ve" means "negative control conditions" which
is the same as experimental conditions but without any active
ingredient (s).
[0064] Preparation of Acetone-Based Application Solutions:
[0065] Acetone--The same 50 .mu.g/.mu.L .omega.-ACTX stock in DMSO
was used to create a formulation in 90% acetone and 10% DMSO that
would deliver a dose equivalent of 90,000 pmol/g when applied as a
2 .mu.L droplet to to houseflies of an average mass of 16 mg. In
this case, the toxin stock was added to the acetone first, and then
a final volume of DMSO was added to reach 10% m/v DMSO. This was
done to examine the amount of precipitate when dissolved toxin was
added to acetone.
[0066] A second .omega.-ACTX solution was also prepared by
dissolving 1.2 mg lyophilized .omega.-ACTX (Auspep) in 240 .mu.L
acetone (5 .mu.g/.mu.L stock). 50 .mu.L of this stock was diluted
in 121.5 .mu.L of Acetone after which 17.15 .mu.L DMSO was added
(10% concentration). Calculations leading to an estimate the
.omega.-ACTX dosage for this formulation are as follows:.
504.times.5 .mu.g/.mu.L .omega.-ACTX stock=250 .mu.g
.omega.-ACTX/171.54 total volume=1.458 .mu.g/.mu.L.times.2
.mu.L/insect=2.915 .mu.g/insect
2.915 .mu.g/insect.times.1 .mu.mol/4050 .mu.g.times.10.sup.6 pmol/1
.mu.mol=719.7 pmol/insect.times.1 insect/0.016 g=45,000 pmol/g
[0067] A control formulation of bovine serum albumin (BSA) was also
prepared in acetone and DMSO. Due to the concentration of the stock
BSA, the concentration of acetone was only about 60% in 10%
DMSO.
TABLE-US-00005 TABLE 3 Acetone-DMSO Treatments .omega.-ACTX DMSO
Dose Concentration Final (pmol/g) (%) .omega.-ACTX DMSO Acetone
Volume 90,000 10% 17.5 .mu.L .omega.-ACTX 12.5 .mu.L 270 .mu.L 300
.mu.L STOCK (50 .mu.g/.mu.L in DMSO) 45,000 10% 50 .mu.L (5
.mu.g/.mu.L in 17.15 .mu.L 104.3 .mu.L 171.5 .mu.L.sup. Acetone)
-ve 10% -- 30 .mu.L 270 .mu.L 300 .mu.L +ve 10% 87.5 .mu.L 10
.mu.g/mL BSA 30 .mu.L 182.5 .mu.L 300 .mu.L
Administration and Application of the Formulation
[0068] Houseflies were refrigerated for .about.4 hr and then
anesthetized with CO.sub.2. Each treatment formulation described
above was applied to a group of ten anesthetized flies. The
treatments consisted of a 2 .mu.L droplet of the respective
formulation, pipetted onto the dorsal thoracic body surface of a
fly. Groups of ten anesthetized flies were used to test each
treatment regime. The Acetone/DMSO solution rapidly evaporated from
the cuticle. The DMSO formulations were allowed to absorb through
the cuticle. Treated flies which revived on their dorsal surface
tended to stick to the bottom of the bin and struggle following
placement with food and water; intervention was made to prevent
this by gently tapping the bin or manipulating stuck flies back to
an upright orientation with tweezers. A control group of untreated
flies was also reserved to ensure mortality was not affected by
CO.sub.2 exposure. All treatments were given food and water and
observed for 24 hours.
[0069] Results (n=10 for all treatment groups, number of dead flies
per group reported in second column):
TABLE-US-00006 TABLE 4 Results of Acetone-DMSO treatments Dead
(Time Treatment (Time Applied) post treatment) Notes -ve 20%
DMSO/Tween 0 (~8 hr) All flies healthy/active (3:54PM Jul. 1, 2008)
90,000 pmol/g .omega.-ACTX 20% DMSO/Tween 1 (~8 hr) 1 dead in food,
others (4:09PM Jul. 1, 2008) healthy 9,000 pmol/g .omega.-ACTX 20%
DMSO/Tween 1 (~7.5 hr) 1 stuck to bottom(?) (4:22PM Jul. 1, 2008)
dead(?) 900 pmol/g .omega.-ACTX 20% DMSO/Tween 0 (~7.5 hr) (4:32PM
Jul. 1, 2008) -ve 10% DMSO/Tween 0 (~6.5 hr) 1 stuck to bottom
& (5:39PM Jul. 1, 2008) dislodged 90,000 pmol/g .omega.-ACTX
10% DMSO/Tween 0 (~7 hr) (4:52PM Jul. 1, 2008) -ve 90% Acetone/10%
DMSO 1 <~7.5 hr) (5:25PM Jul. 1, 2008) +ve BSA Protein 1(?) (~7
hr) 1 unresponsive on side (5:01PM Jul. 1, 2008) of food dish
90,0000 pmol/g .omega.-ACTX (DMSO Stock) 0 (~7 hr) 1 stuck on back
& 90%Acetone/10% DMSO (5:11PM Jul. 1, 2008) dislodged 45,0000
pmol/g .omega.-ACTX (Acetone Stock) 1 (~6.5 hr) 1 dead in food dish
90%Acetone/10% DMSO (5:19PM Jul. 1, 2008) -ve untreated (5:40PM
Jul. 1, 2008) 0 (~6.5 hr) -ve 20% DMSO/Tween 0 (~19 hr) All flies
healthy/active (3:54PM Jul. 1, 2008) 90,000 pmol/g .omega.-ACTX 20%
DMSO/Tween 1 (~19 hr) 1 twitching (4:09PM Jul. 1, 2008) 9,000
pmol/g .omega.-ACTX 20% DMSO/Tween 1 (~18.5 hr) Dead from sticking
to (4:22PM Jul. 1, 2008) bottom 900 pmol/g .omega.-ACTX 20%
DMSO/Tween 0 (~18.5 hr) All flies healthy/active (4:32PM Jul. 1,
2008) -ve 10% DMSO/Tween 1 (~17.5 hr) Dead in food (5:39PM Jul. 1,
2008) 90,000 pmol/g .omega.-ACTX 10% DMSO/Tween 1 (18 hr) 2
twitching (4:52PM Jul. 1, 2008) -ve 90% Acetone/10%DMSO (5:25PM
Jul. 1, 2008) 1 (~17.5 hr) +ve BSA Protein (5:01PM Jul. 1, 2008) 1
(~18 hr) 90,0000 pmol/g .omega.-ACTX (DMSO Stock) 1 (~18 hr) 1
twitching 90% Acetone/10% DMSO (5:11PM Jul. 1, 2008) 45,0000 pmol/g
.omega.-ACTX (Acetone Stock) 5 (~17.5 hr) 2 twitching
90%Acetone/10% DMSO (5:19PM Jul. 1, 2008) -ve untreated (5:40PM
Jul. 1, 2008) 0 (~17.5 hr) All flies healthy/active Dead (Time
Treatment post treatment) Notes -ve 20% DMSO/Tween 0 (~27.5 hr)
(3:54PM Jul. 1, 2008) 90,000 pmol/g .omega.-ACTX 20% DMSO/Tween 3
(~27.5 hr) 1 twitching (4:09PM Jul. 1, 2008) 9,000 pmol/g
.omega.-ACTX 20% DMSO/Tween 1 (~27 hr) (4:22PM Jul. 1, 2008) 900
pmol/g .omega.-ACTX 20% DMSO/Tween 0 (~27 hr) (4:32PM Jul. 1, 2008)
-ve 10% DMSO/Tween 1 (~26 hr) (5:39PM Jul. 1, 2008) 90,000 pmol/g
.omega.-ACTX 10% DMSO/Tween 3 (~26.5 hr) 1 twitching (4:52PM Jul.
1, 2008) -ve 90% Acetone/10%DMSO 1 (~26 hr) (5:25PM Jul. 1, 2008)
+ve BSA Protein (5:01PM Jul. 1, 2008) 1 (~26 hr) 90,0000 pmol/g
.omega.-ACTX (DMSO Stock) 3 (~26.5 hr) 1 twitching 90%Acetone/10%
DMSO (5:11PM Jul. 1, 2008) 45,0000 pmol/g .omega.-ACTX (Acetone
Stock) 7 (~26 hr) 1 sick 90%Acetone/10% DMSO (5:19PM Jul. 1, 2008)
-ve untreated (5:40PM Jul. 1, 2008) 0 (~26 hr)
[0070] Topical application of .omega.-ACTX dissolved in Acetone
with 10% DMSO was insecticidal to flies (70% mortality at 24 hrs.)
while a similar preparation of .omega.-ACTX dissolved in DMSO then
diluted to 10% DMSO in acetone was less insecticidal (.about.30%).
These results are similar to the topical assays in which
.omega.-ACTX dissolved in Acetone with DMSO added to a
concentration of 10% killed 90% of houseflies (Jun. 19, 2008) while
.omega.-ACTX dissolved in DMSO and then diluted in Acetone to a
concentration of 90,000 pmol/g killed 40% of treated insects.
Topical application of .omega.-ACTX in 10-20% DMSO in water was
also insecticidal, but considerably less so than the Acetone/DMSO
solution (30% vs. 70%).
Example 2
Topical Assay with Acetone/Methanol/DMSO using House Fly
[0071] Toxin is .omega.-ACTX-Hv1a:
TABLE-US-00007 (SEQ. ID. NO. 60)
SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD
[0072] Synthetic. Molecular weight: 4050 Da. LD.sub.50 in
House-fly: 90.2 pmol/g.
[0073] Administration and application of the formulation
[0074] Insects: House fly (Musca domestica) from Benzon research
weighing between 12-18 mg (average mass 15 mg); 2 .mu.L
micropipette application onto dorsal thorax.
[0075] Cabbage Looper (Trichoplusia ni) from Benzon research
weighing .about.30 mg; 2 .mu.L micropipette application to dorsal
anterior.
[0076] Toxin Dose Calculations: .about.90,000 pmol/g .omega.-ACTX
(1000.times. Injection LD.sub.50),
[0077] 0.015 g/fly.times.90,000 pmol/g=1350 pmol/fly.times.4050
.mu.g/pmol.times.1 .mu.g/10.sup.6 pg=5.467 .mu.g/insect.
[0078] 5.467 .mu.g/2 .mu.L application=2.733 .mu.g/.mu.L.times.150
.mu.L=410.06 .mu.g.times.1 .mu.L/5 .mu.g=82 L 5 .mu.g/.mu.L
.omega.-ACTX stock
Preparation of Application Solutions.
[0079] Mixtures of .omega.-ACTX in acetone (90%) and DMSO (10%)
were prepared according to Table 5 from a stock preparation of 1.5
mg lyophilized .omega.-ACTX dissolved in 300 .mu.L of acetone to
produce a 5 mg/mL solution. .omega.-ACTX formed a cloudy
precipitate when acetone was added which settled out when left on
the bench. The preparation was vortexed for .about.5 sec to
homogenize the precipitate prior to dilution.
[0080] Methanol/DMSO--Mixtures of .omega.-ACTX in methanol (90%)
and DMSO (10%) were prepared according to Table 5 from a stock
preparation of 2.3 mg lyophilized .omega.-ACTX dissolved in 460
.mu.L of methanol to produce a mixture with a final peptide
concentration of 5 mg/mL. .omega.-ACTX formed a cloudy precipitate
when methanol was added which settled out when left on the bench,
in a similar manner to atracotoxin/acetone suspensions. The
preparation was vortexed for .about.5 sec. to homogenize the
precipitate prior to dilution.
TABLE-US-00008 TABLE 5 Treatment Preparations. Total Treatment
.omega.-ACTX DMSO Solvent Volume 90,000 82 .mu.L 5 mg/mL 15 .mu.L
53 .mu.L 150 .mu.L pmol/g .omega.-ACTX Acetone STOCK in Acetone -ve
-- 15 .mu.L 135 .mu.L 150 .mu.L Acetone 90,000 82 .mu.L 5 mg/mL 15
.mu.L 53 .mu.L 150 .mu.L pmol/g .omega.-ACTX Methanol STOCK in
Methanol -ve -- 15 .mu.L 135 .mu.L 150 .mu.L Methanol
[0081] Table 5 treatment preparations are are formulations of
acetone/methanol/DMSO; order of addition when preparing each
formulation was solvent, .omega.-ACTX stock (where necessary), and
finally DMSO.
Administration and Application of the Formulation.
[0082] Each treatment formulation described above was applied to a
group of ten CO.sub.2-anesthetized houseflies. Treatment
application consisted of a 2 .mu.L droplet of the respective
formulation, pipetted onto the dorsal thoracic body surface of a
fly. Each mixture was vortexed immediately prior to each
application to ensure suspension of precipitate particles.
Following treatment, insects were placed in bins with fresh food
and water, allowed to recover, and observed over 24 hours.
[0083] Each treatment formulation described above was also applied
to a group of ten 2.sup.nd instar T. ni. Treatment application
consisted of a 2 .mu.L droplet of the respective formulation,
pipetted onto the anterior dorsal body surface. Each mixture was
vortexed and immediately prior to each application to ensure
suspension of precipitate particles. All treatment mixtures were
vortexed immediately prior application to ensure suspension of
precipitate particles. Following treatment, insects were placed on
fresh media and observed for 24 hrs.
TABLE-US-00009 TABLE 6a Housefly Dead Dead Treatment (18 hrs.) (24
hrs.) 90,000 pmol/g Acetone + 4 4 DMSO -ve Acetone + DMSO 0 0
90,000 pmol/g Methanol + 10 10 DMSO -ve Methanol + DMSO 0 0
Untreated 0 0
TABLE-US-00010 TABLE 6b Cabbage Looper Treatment Dead (18 hr) Dead
(24 hr) 45,000 pmol/g Acetone + 0 0 DMSO -ve Acetone + DMSO 0 0
45,000 pmol/g Methanol + 0 0 DMSO -ve Methanol + DMSO 0 0
[0084] Table 6a and Table 6b (above) Results of administration of
acetone/methanol/DMSO formulations to Housefly and Cabbage
Looper.
[0085] Topical treatment of houseflies with a high dose of
.omega.-ACTX in methanol with DMSO was insecticidal with 100%
mortality at 18 hours post treatment compared to only 40% mortality
of houseflies treated with .omega.-ACTX in acetone. There was no
control mortality in either treatment. There was no difference
between .omega.-ACTX and control treatments of cabbage loopers in
terms of insect death, feeding, or behavior. Methanol potentiated
the topical activity of .omega.-ACTX more than acetone in this
experiment.
Example 3
Topical Assay with Methanol and Ethanol using Housefly
[0086] Toxin is .omega.-ACTX-Hv1a
TABLE-US-00011 (SEQ ID. NO: 60)
SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD
[0087] Synthetic. Molecular weight: 4050 Da
[0088] LD.sub.50 in House-fly: 90.2 pmol/g
Administration and Application of the Formulation
[0089] Insect: House fly (Musca domestica) from Benzon research
weighing between 12-20 mg (average mass 16 mg). 2 .mu.L
micropipette application onto dorsal thorax.
[0090] Toxin Dose Calculations:
[0091] .about.90,000 pmol/g .omega.-ACTX (1000.times. Injection
LD.sub.50),
[0092] 0.015 g/fly.times.90,000 pmol/g=1350 pmol/fly.times.4050
pg/pmol.times.1 .mu.g/10.sup.6 pg=5.467 .mu.g/insect
[0093] 5.467 .mu.g/2 .mu.L application=2.733 .mu.g/.mu.L.times.250
.mu.L=683.43 .mu.g.times.1 .mu.L/5 .mu.g=136.7 .mu.L 5 .mu.g/.mu.L
.omega.-ACTX stock
[0094] NOTE: Treatment solutions calculated for flies of an average
mass of 15 mg (12-18 mg range); actual average mass of insects used
was 16 mg (12-20 mg range). .omega.-ACTX dose in tables below has
been adjusted for this discrepancy.
Preparation of Application Solutions
[0095] Methanol--Solutions of .omega.-ACTX in Methanol were
prepared according to Table 7, using a stock preparation of 1.0 mg
lyophilized .omega.-ACTX dissolved in 200 .mu.L of methanol (5
mg/mL solution).
TABLE-US-00012 TABLE 7 Methanol Treatment Formulations Total
Treatment .omega.-ACTX Methanol Volume 84,375 pmol/g 136.68 .mu.L 5
mg/mL .omega.-ACTX stock 113.3 .mu.L.sup. 250 .mu.L 16,875 pmol/g
50 .mu.L 84,375 pmol/g solution 200 .mu.L 250 .mu.L 3,375 pmol/g 50
.mu.L 16,875 pmol/g solution 200 .mu.L 250 .mu.L 675 pmol/g 50
.mu.L 3,375 pmol/g solution 200 .mu.L 250 .mu.L 135 pmol/g 50 .mu.L
675 pmol/g solution 200 .mu.L 250 .mu.L -ve -- 250 .mu.L 200
.mu.L
[0096] Methanol+DMSO--Solutions of .omega.-ACTX in methanol were
prepared according to Table 8 using a 5 mg/mL stock. The 84,375
pmol/g solution was prepared by diluting the stock mixture into
methanol (88.3 .mu.L) before adding DMSO (25 .mu.L) to a final
concentration of 10% DMSO. A 10% DMSO in methanol solution was then
prepared and aliquoted as described below for the serial dilution
series.
TABLE-US-00013 TABLE 8 Methanol/DMSO Treatment Solutions Total
Treatment .omega.-ACTX DMSO Methanol Volume 84,375 pmol/g 136.68
.mu.L 5 mg/mL 25 .mu.L 88.3 .mu.L 250 .mu.L .omega.-ACTX STOCK in
Methanol (100%) 16,875 pmol/g 50 .mu.L 84,375 pmol/g solution --
200 .mu.L 250 .mu.L (10% DMSO) 3,375 pmol/g 50 .mu.L 16,875 pmol/g
solution -- 200 .mu.L 250 .mu.L (10% DMSO) 675 pmol/g 50 .mu.L
3,375 pmol/g solution -- 200 .mu.L 250 .mu.L (10% DMSO) 135 pmol/g
50 .mu.L 675 pmol/g solution -- 200 .mu.L 250 .mu.L (10% DMSO) -ve
-- -- 250 .mu.L 250 .mu.L (10% DMSO)
[0097] Ethanol+DMSO--Solutions of .omega.-ACTX in Ethanol were
prepared according to Table 9 from a stock preparation of 0.8 mg
lyophilized .omega.-ACTX dissolved in 160 .mu.L of Ethanol to
produce a 5 mg/mL solution. The 84,375 pmol/g solution was prepared
by diluting the stock solution into ethanol (88.3 .mu.L) before
adding DMSO (25 .mu.L) to a final concentration of 10% DMSO. A 10%
DMSO/ethanol solution was then prepared and aliquoted as described
below for the serial dilution series.
TABLE-US-00014 TABLE 9 Ethanol Solution Formulations; order of
addition when preparing the 84,375 pmol/g formulation was ethanol,
.omega.-ACTX stock and finally DMSO. A 10% DMSO/Ethanol solution
was prepared and aliquoted in labeled tubes, and a 5x serial
dilution from the 84,375 pmol/g treatment was carried out. Total
Treatment .omega.-ACTX DMSO Ethanol Volume 84,375 pmol/g 136.68
.mu.L 5 mg/mL .omega.-ACTX in 25 .mu.L 88.3 .mu.L 250 .mu.L Ethanol
(100%) 16,875 pmol/g 50 .mu.L 84,375 pmol/g solution -- 200 .mu.L
250 .mu.L (10% DMSO) 3,375 pmol/g 50 .mu.L 16,875 pmol/g solution
-- 200 .mu.L 250 .mu.L (10% DMSO) 675 pmol/g 50 .mu.L 3,375 pmol/g
solution -- 200 .mu.L 250 .mu.L (10% DMSO) 135 pmol/g 50 .mu.L 675
pmol/g solution -- 200 .mu.L 250 .mu.L (10% DMSO) -ve -- -- 250
.mu.L 250 .mu.L (10% DMSO)
Administration and Application of the Formulation
[0098] Each treatment formulation described above was applied to a
group of ten CO.sub.2-anesthetized houseflies. Treatment
application consisted of a 2 .mu.L droplet of the respective
formulation, pipetted onto the dorsal thoracic body surface of a
fly. Each mixture was vortexed immediately prior to each
application to ensure suspension of precipitate particles.
Following treatment, insects were placed in bins with fresh food
and water, allowed to recover, and observed over 60 hours.
Results of Methanol or Ethanol and DMSO Treatments
TABLE-US-00015 [0099] TABLE 10 Results of treatment with methanol
or ethanol and DMSO Dead Dead Dead Dead Dead Treatment (6 hrs.)
(19.5 hrs.) (24 hrs.) (50 hrs.) (60 hrs.) -ve control Methanol +
DMSO 0 0 0 0 0 135 pmol/g Methanol + DMSO 0 0 0 0 0 675 pmol/g
Methanol + DMSO 0 0 0 0 0 3,375 pmol/g Methanol + DMSO 0 0 0 0 0 (1
twitch) 16,875 pmol/g Methanol + DMSO 0 0 0 0 0 (1 twch) (1 twch)
(1 twch) 84,375 pmol/g Methanol + DMSO 0 3 3 7 7 (3 twch) (3 twch)
(1 twitch) -ve control Methanol 0 0 0 0 0 135 pmol/g Methanol 0 0 0
0 0 675 pmol/g Methanol 0 0 0 0 0 3,375 pmol/g Methanol 0 0 0 0 0
16,875 pmol/g Methanol 0 0 0 1 1 (1 twch) 84,375 pmol/g Methanol 0
0 0 1 1 (5 twch) -ve control Ethanol + DMSO 0 0 0 0 0 135 pmol/g
Ethanol + DMSO 0 0 0 0 0 675 pmol/g Ethanol + DMSO 1 1 1 1 1 3,375
pmol/g Ethanol + DMSO 0 0 0 1 0 16,875 pmol/g Ethanol + DMSO 0 3 3
7 7 (1 twch) (2 twch) (1 twch) 84,375 pmol/g Ethanol + DMSO 2 7 7 7
7 (1 twch) Untreated 0 0 0 0 0
[0100] Topical treatment of houseflies with a high dose (84,375
pmol/g) of .omega.-ACTX in ethanol with DMSO was insecticidal
causing 30% and 70% mortality at 24 and 50 hours post treatment,
respectively. The treatments prepared with ethanol were more potent
than those prepared with methanol (70% vs. 30% mortality at 24 hrs.
for the highest dose, and 30% vs. 0% mortality in 16,875 pmol/g
treatment at 24 hrs.). The effect of the highest dose of
.omega.-ACTX in methanol with DMSO was not as potent as in the
previous assay (3 dead, 3 twitching vs. 10 dead at the highest dose
after 24 hrs.).
[0101] Methanol/.omega.-ACTX treatments without DMSO were not
insecticidal, suggesting inclusion of an aprotic penetrant or some
other type of molecular adjuvant is important to the activity of
topical preparations of .omega.-ACTX.
[0102] This experiment shows examples of effective dose ranges of
topically applied .omega.-ACTX diluted in methanol, and what effect
the inclusion of DMSO and ethanol has on the insecticidal activity
of the formulation in the topical bioassay paradigm used.
Example 4
[0103] Topical Assay with the toxin: .omega.-ACTX-Hv1a:
TABLE-US-00016 (SEQ ID. NO. 55.)
SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD
[0104] Molecular weight: 4050. LD.sub.50 in House-fly: 90.2
pmol/g
[0105] Freeze dried aliquots of 1.5 mg toxin prepared from frozen
stocks
[0106] Topical application: groups of ten house flies (Musca
domestica) from Benzon research, weighing between 12-20 mg (average
mass 16 mg), each received a 2 .mu.L micropipette application of
toxin precipitate suspended in Ethanol-DMSO on the dorsal thoracic
surface of the body.
[0107] Preparation of stock solutions for topical and per os
treatment:
[0108] Stock 1 (vortexed preparation): 1 mL ethanol was added to
.about.1500 .mu.g lyophilized .omega.-ACTX, and the resulting
mixture vortexed vigorously. A 50 .mu.L aliquot of the peptide
suspension was then removed for topical application assays and kept
on ice for .about.2 hrs.; the remainder was divided into two
.about.475 .mu.L aliquots and then kept on ice for .about.2
hrs.
[0109] Stock 2 (vortexed and sonicated preparation): 1 mL ethanol
was added to .about.1500 .mu.g lyophilized .omega.-ACTX, and the
resulting mixture vortexed vigorously, and then sonicated
.about.10-15 sec., gently ramping up from intensity setting "0" to
setting "5" during this period. A 50 .mu.L aliquot of the peptide
suspension was then removed for topical application assays and kept
on ice for .about.2 hrs.; the remainder was divided into two
.about.475 .mu.L aliquots and then kept on ice for .about.2
hrs.
[0110] Toxin Dose Calculations:
[0111] 1.5 .mu.g/.mu.L.times.2 .mu.L/application.times.10.sup.6
pg/1 .mu.g.times.1 pmol/4050 pg.times.1 fly/0.016 g=46,875
pmol/g
Topical Application of Toxin Solutions and Results Thereof
[0112] Stock solutions of .omega.-ACTX in Ethanol were prepared as
described above. Table 11, below, indicates the recipes used to
dilute the stock solutions for topical application to
houseflies:
TABLE-US-00017 TABLE 11 Ethanol/DMSO formulations Treatment# 90%
EtOH/ Total (dose) .omega.-ACTX DMSO 10% DMSO Volume 1-vortexed 50
.mu.L .omega.-ACTX Stock-1 5 .mu.L -- 55 .mu.L (46,875 pmol/g)
2-vortexed 10 .mu.L treatment 1 solution -- 40 .mu.L 50 .mu.L
(9,375 pmol/g) 3-vortexed + 50 .mu.L .omega.-ACTX Stock-2 5 .mu.L
-- 55 .mu.L sonicated (46,875 pmol/g) 4- vortexed + 10 .mu.L
treatment 3 solution -- 40 .mu.L 50 .mu.L sonicated (9,375
pmol/g)
[0113] Treated flies were kept in containers with ad libitum access
to food and water and mortality (and "twitching" behavior,
presumably resulting from disruption of physiological norms by
action of the toxin) was scored thereafter as indicated in Table 12
below:
TABLE-US-00018 TABLE 12 Results of treament with Ethanol/DMSO Dead
Dead Dead Dead Treatment N (7.5 hr) (14 hr) Dead (24 hr) (40 hr)
(48 hr) Dead (76 hr) 9,000 pmol/g 10 0 1 1 2 2 3 vortexed (1 twch)
(1 twch) (2 twch) (1 twch) 45,000 pmol/g 10 1 6 6 8 8 8 vortexed (2
twitch) (1 twch) 9,000 pmol/g 10 2 2 2 3 3 3 sonicated (2 twch) (1
twch) (2 twch) (1 twch) 45,000 pmol/g 10 5 8 9 9 9 9 sonicated (2
twch)
[0114] This experiment demonstrates that sonicating .omega.-ACTX
suspended in ethanol increases the topical insecticidal activity of
the resulting toxin formulation.
[0115] The sonication of ethanol-DMSO precipitates of
omega-ACTX-Hv1a enhances the insecticidal activity of the omega
toxin by increasing the mortality of contact-treated houseflies, up
to 24 hrs. post application.
Example 5
Toxins are:
[0116] 1) .omega.-ACTX-Hv1a+2:
TABLE-US-00019 (SEQ ID. NO. 117) GSSPT CIPSG QPCPY NENCC SQSCT
FKENE NGNTV KRCD
has three disulfide bridges: 6-20, 13-24 and 19-38.
[0117] Molecular weight: 4199. Injection LD50 in Housefly: 77
pmol/g
[0118] rKappa-ACTX-Hv1c:
TABLE-US-00020 SEQ ID. NO. 118 GSAIC TGADR PCAAC CPCCP GTSCK AESNG
VSYCR KDEP
has four disulfide bridges: 5-19, 12-24, 15-16, 18-34
[0119] Recombinant from pDR2 (pET-32a). Molecular weight:
3912.15
[0120] Injection LD50 in Housefly: 389 pmol/g
[0121] rU-ACTX-Hv1a+2:
TABLE-US-00021 SEQ ID. NO. 119 GSQYC VPVDQ PCSLN TQPCC DDATC TQERN
ENGHT VYYCR A
has three disulfide bridges: 5-20, 12-25, 19-39
[0122] Molecular Weight: 4570.51
[0123] Injection LD50 in Housefly: 81.5 pmol/g
[0124] Preparation of mixtures for topical treatment:
Recipes for toxin stocks used to formulate treatment mixtures were
as follows: Stock 1: .omega.-ACTX-Hv1a+2: an aliquot of 1.5 mg
freeze dried toxin suspended in 850 .mu.L ethanol and sonicated for
10-15 seconds with gentle ramp from setting "0" to setting "5" on
sonicator to create fine particles. Stock 2: rU-ACTX-Hv1a+2 an
aliquot of 1.5 mg freeze dried toxin suspended in 900 .mu.L acetone
and sonicated for 10-15 seconds with gentle ramp from setting "0"
to setting "5" on sonicator to create fine particles. Stock 3:
rU-ACTX-Hv1a+2: an aliquot of 1.5 mg freeze dried toxin suspended
in 900 .mu.L methanol and sonicated for 10-15 seconds with gentle
ramp from setting "0" to setting "5" on sonicator to create fine
particles. Stock 4: rKappa-Hv1c+2: an aliquot of 1.5 mg freeze
dried toxin suspended in 900 .mu.L acetone and sonicated for 10-15
seconds with gentle ramp from setting "0" to setting "5" on
sonicator to create fine particles. Stock 5: rKappa-Hv1c+2: an
aliquot of 1.5 mg freeze dried toxin suspended in 900 .mu.L
methanol and sonicated for 10-15 seconds with gentle ramp from
setting "0" to setting "5" on sonicator to create fine
particles.
[0125] Final preparation of mixtures for topical applications was
done by mixing stocks with other reagents as listed below:
Control 1--Ethanol+0.05% LI-700-475 .mu.L Ethanol. 25 .mu.L 1%
LI-700 in Ethanol
Control 2--Ethanol+0.01% LI-700-495 .mu.L Ethanol. 5 .mu.L 1%
LI-700 in Ethanol
Control 3--Ethanol+0.1% MSO.RTM.-450 .mu.L Ethanol. 50 .mu.L 1%
MSO.RTM. in Ethanol
Control 4--Ethanol+0.02% MSO.RTM.-490 .mu.L Ethanol . 10 .mu.L 1%
MSO.RTM. in Ethanol
Treatment 1--.omega.-ACTX-Hv1a+2 Ethanol Precipitate+10% DMSO+0.05%
Silwet-425 .mu.L
Stock 1
25 .mu.L 1% Silwet in Ethanol. 504 DMSO
Control 5--Ethanol+10% DMSO+0.05% Silwet-425 .mu.L Ethanol
25 .mu.L 1% Silwet in Ethanol. 504 DMSO
[0126] Treatment 2--rU-ACTX-Hv1a+2 acetone precipitate+10% DMSO-450
.mu.L Stock 2. 50 .mu.L
DMSO
[0127] Treatment 3--rU-ACTX-Hv1a+2 methanol precipitate+10%
DMSO-450 .mu.L Stock 3. 50 .mu.L
DMSO
[0128] Treatment 4--rKappa-ACTX-Hv1c acetone precipitate+10%
DMSO-450 .mu.L Stock 4.
50 .mu.L DMSO
[0129] Treatment 5--rKappa-ACTX-Hv1c methanol precipitate+10%
DMSO-450 .mu.L Stock 5.
50 .mu.L DMSO
Control 6--Acetone+10% DMSO-450 .mu.L Acetone. 50 .mu.L DMSO
[0130] Control 7--Methanol+10% DMSO-450 .mu.L methanol. 50 .mu.L
DMSO
Control 8--Ethanol+10% DMSO-450 .mu.L Ethanol. 50 .mu.L DMSO
Administration and Application of the Formulation.
[0131] Topical application of 24 droplets to the ventral abdomen of
houseflies between 12-18 mg with P10 micropipettes, as described in
previous examples. After application, flies were provided food and
water ad libitum and observed for mortality.
[0132] Results of the mixtures of topical treatments. Table 13
(below.)
TABLE-US-00022 6 hrs. 24 hrs. 42 hrs. Treatment Twitch/ Twitch/
Twitch/ LI-700 (n = 10) Dead Morib Dead Morib Dead Morib Control 1
- Ethanol + 0.05% LI-700 0 4/0 0 3/1 1 1/1 Control 2 - Ethanol +
0.01% LI-700 0 6/0 1 4/1 1 4/2 MSO (n = 10) Control 3 - Ethanol +
0.1% MSO .RTM. 2 4/1 2 4/0 3 2/1 Control 4 - Ethanol + 0.02% 0 7/0
1 5/0 4 1/1 MSO .RTM. Silwet (n = 10, ~45,000 pmol/g) Treatment
1-.omega.-ACTX-Hv1a + 2 2 3/0 7 1/2 10 0/0 precipitate + ~10% DMSO
+ 0.05% Silwet Control 5 - Ethanol + 1 0/0 1 0/0 2 0/0 ~10% DMSO +
0.05% Silwet Acetone/Methanol Precipitation (n = 10, ~45,000
pmol/g) Treatment 2 - rU-ACTX-Hv1a + 2 0 0/0 1 2/0 5 2/0 acetone
precipitate + 10% DMSO Treatment 3 - rU-ACTX-Hv1a + 2 0 0/0 2 1/0 5
2/0 methanol precipitate + 10% DMSO Treatment 4 - rKappa-ACTX- 0
1/0 0 1/0 5 3/0 Hv1c + 2 acetone precipitate* + 10% DMSO Treatment
5 - rKappa-ACTX- 0 1/0 1 1/0 4 0/0 Hv1c + 2 methanol precipitate* +
10% DMSO Control 6 - Acetone + 10% DMSO 0 0/0 1 0/0 2 0/0 Control 7
- Methanol + 10% 0 0/0 0 0/0 1 0/0 DMSO Control 8 - Ethanol + 10%
DMSO 0 0/0 0 0/0 0 0/0
[0133] Concentrations of LI-700 down to 0.01% resulted in
considerable disruption in the behavior of the treated flies, and
possibly some mortality as well. Concentrations of MSO.RTM. down to
0.02% resulted in considerable disruption in the behavior of the
treated flies, and considerable (i.e., 30-40%) mortality as well.
Silwet, 0.05%., may slightly potentiate the topical insecticidal
activity of omega-toxin/ethanol/dmso suspensions in this
experimental paradigm. Based on results presented here and other
undisclosed studies potentiation would be in the range of
15-20%.
[0134] Topically applied formulations of the hybrid and kappa
atracotoxin-1s are insecticidal when 90% acetone or 90% methanol is
substituted for 90% ethanol. The acetone and methanol formulations
of the hybrid toxin may be slightly less insecticidal than the
previously tested ethanol formulation. We believe that acetone and
methanol formulations result in equivalent or slightly higher
levels of insecticidal activity when compared to ethanol
formulations of kappa toxin.
Example 6
[0135] Toxin to Apply Topically is .omega.-ACTX-Hv1a:
TABLE-US-00023 (SEQ ID. NO. 60)
SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD
having a Molecular weight: 4050. LD50 in House-fly: 90.2
pmol/g.
[0136] Freeze dried aliquots of 1.5 mg toxin prepared from frozen
stocks.
Administration and Application of the Formulation
[0137] Topical application: groups of ten house flies (Musca
domestica) from Benzon research, weighing between 12-20 mg (average
mass 16 mg), each received a 2 .mu.L micropipette application of
toxin precipitate suspended in Ethanol-DMSO on the dorsal thoracic
surface of the body.
Preparation of Stock Solutions for Tropical Treatment:
[0138] Stock 1 (vortexed and sonicated Ethanol preparation): 0.9 mL
ethanol was added to .about.1500 .mu.g lyophilized .omega.-ACTX,
and the resulting mixture vortexed vigorously, and then sonicated
.about.10-15 sec., gently ramping up from intensity setting "0" to
setting "5" during this period. 0.1 mL DMSO was then added to the
toxin suspension, the resulting mixture was vortexed, and a 100
.mu.L aliquot of the alcohol-DMSO-peptide suspension was then
removed for topical application assays and kept on ice for .about.2
hrs.
[0139] Stock 2 (vortexed and sonicated 1-Propanol preparation): 0.9
mL 1-propanol was added to .about.1500 .mu.g lyophilized
.omega.-ACTX, and the resulting mixture vortexed vigorously, and
then sonicated .about.10-15 sec., gently ramping up from intensity
setting "0" to setting "5" during this period. 0.1 mL DMSO was then
added to the toxin suspension, the resulting mixture was vortexed,
and a 100 .mu.L aliquot of the alchohol-DMSO-peptide suspension was
then removed for topical application assays and kept on ice for
.about.2 hrs.
[0140] Stock 3 (vortexed and sonicated 2-Propanol preparation): 0.9
mL 2-propanol was added to .about.1500 .mu.g lyophilized
.omega.-ACTX, and the resulting mixture vortexed vigorously, and
then sonicated .about.10-15 sec., gently ramping up from intensity
setting "0" to setting "5" during this period. 0.1 mL DMSO was then
added to the toxin suspension, the resulting mixture was vortexed,
and a 100 .mu.L aliquot of the alcohol-DMSO-peptide suspension was
then removed for topical application assays and kept on ice for
.about.2 hrs.
[0141] Stock 4 (vortexed and sonicated 2-Butanol preparation): 0.9
mL 2-butanol was added to .about.1500 .mu.g lyophilized
.omega.-ACTX, and the resulting mixture vortexed vigorously, and
then sonicated .about.10-15 sec., gently ramping up from intensity
setting "0" to setting "5" during this period. 0.1 mL DMSO was then
added to the toxin suspension, the resulting mixture was vortexed,
and a 100 .mu.L aliquot of the alcohol-DMSO-peptide suspension was
then removed for topical application assays and kept on ice for
.about.2 hrs.
[0142] Note that each stock was made to a concentration such that a
2 .mu.L application of the stock to the body surface of a .about.16
mg housefly would result in a toxin dose of .about.45,000 pmol/g.
Hence, in some cases described below, one of the four stocks
described above was topically applied full-strength to houseflies,
but in other cases, five-fold serial dilutions were performed
(using the stocks and the corresponding 90% alcohol-10% DMSO
solution) in order to obtain a solution that could be used to
deliver a lower toxin dose in a 2 .infin.L volume. Negative control
procedures (indicated as "-ve" in the table below) comprised house
flies treated with 2 .mu.L dorsal throracic applications of
solutions of the alcohols in question (diluted to 90% v/v with
DMSO).
Topical Application of Toxin Solutions and Results Thereof:
[0143] Stock solutions of .omega.-ACTX in various alcohols were
prepared as described above. Table 14 below indicates the stock
formulations and dosing used for topical application to houseflies
and the observed mortality for the corresponding groups of
flies:
TABLE-US-00024 TABLE 14 Results of topical application of toxin
solutions # of Flies Dead Dead Dead Dead Treatment Treated (~16 hr)
(24 hr) (52 hr) (64 hr) 1-Propanol -ve 10 0 0 0 0 1-Propanol 10 0 0
1 1 9,000 pmol/g 1-Propanol 10 1 2 2 2 45,000 pmol/g (1 twitch) (1
twitch) (1 twitch) (1 twitch) 2-Propanol -ve 10 1 2 3 3 (2 twitch)
(1 twitch) 2-Propanol 10 3 4 4 4 9,000 pmol/g (2 twitch) (1 twitch)
2-Propanol 10 5 5 8 8 45,000 pmol/g (3 twitch) (3 twitch) 2-Butanol
-ve 10 5 7 7 7 2-Butanol 10 6 6 6 7 9,000 pmol/g (1 twitch) (1
twitch) (2 twitch) Ethanol -ve 7 0 0 0 0 Ethanol 10 0 1 1 1 1,800
pmol/g Ethanol 10 2 2 2 2 9,000 pmol/g (2 twitch) 1-Octanol -ve 10
10 10 10 10
When normalized for mortality observed in negative control groups
(dosed with the corresponding alcohol-DMSO solution), ethanol
precipitates of omega toxin appear to have insecticidal activity as
high or higher than any other toxin-alcohol precipitate tested in
this series of experiments.
[0144] 90% octanol-10% DMSO, 90% 2-butanol-10% DMSO, and 90%
2-propanol-10% DMSO appear to cause unacceptable levels of
background mortality; the latter could presumably mask mortality
due to target site action of omega toxin in treated houseflies. 90%
1-propanol-10% DMSO does not appear to cause unacceptable levels of
background mortality, but it also does not appear to potentiate
target site activity of applied toxin as well as 90% ethanol-10%
DMSO.
Example 7
Toxin to Apply Topically
TABLE-US-00025 [0145] .omega.-ACTX-Hv1a+2: (SEQ ID. NO. 117)
GSSPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD.
[0146] Molecular weight: 4196
[0147] Freeze dried aliquots of 1.5 mg toxin prepared from frozen
stocks
Treatment Mixture Preparation
[0148] All treatments were made to 1.5 .mu.g/.mu.L final
concentration of .omega.-ACTX-Hv1a+2. As previously, ethanol was
added to .about.1500 .mu.g samples of lyophilized
.omega.-ACTX-Hv1a+2, the resulting mixture was vortexed vigorously,
then sonicated .about.10-15 sec., gently ramping up from intensity
setting "0" to setting "5" during this period. Additional
ingredients such as DMSO, MSO.RTM., water, and Tween 20 detergent
were added following sonication, and the resulting mixtures were
vortexed vigorously prior to topical application in order to ensure
even mixing of ingredients. Assuming average fly mass of 16 mg
(12-20 mg cohort) dosing is calculated as follows:
3 .mu.g/insect.times.1 .mu.mol//4196 .mu.g.times.10.sup.6 pmol/1
.mu.mol.times.1 insect/0.016 g=44,685 pmol/g dose
[0149] Recipes for toxin stocks used to formulate treatment
mixtures were as follows:
Stock 1--1.5 mg lyophilized .omega.-ACTX-Hv1a+2 dissolved in 500
.mu.L ethanol, vortexed and sonicated. Stock 2--1.5 mg lyophilized
.omega.-ACTX-Hv1a+2 dissolved in 150 .mu.L sterile water (10
mg/mL).
[0150] Topical Assays using .omega.-ACTX-Hv1a+2 with DMSO:
Recipes for treatment mixtures were as follows: 45,000 pmol/g
.omega.-ACTX-Hv1a+2 90% Ethanol/10% DMSO solution (+-ve control)-50
.mu.L Stock 1, 40 .mu.L ethanol, 10 .mu.L DMSO. 90% Water/10%
DMSO/0.1% Tween 20 eve control)--7.5 .mu.L Stock 2, 82.5 .mu.L
sterile water, 10 .mu.L DMSO. 1 .mu.L 10% Tween 20. 45,000 pmol/g
.omega.-ACTX-Hv1a+2 80% Ethanol/10% Water/10% DMSO-50 .mu.L Stock
1, 30 .mu.L ethanol, 10 gL sterile water, 10 .mu.L DMSO. 45,000
pmol/g .omega.-ACTX-Hv1a+2 70% Ethanol/20% Water/10% DMSO-50 .mu.L
Stock 1, 20 .mu.L ethanol, 20 gL sterile water, 10 .mu.L DMSO.
45,000pmol/g .omega.-ACTX-Hv1a+2 60% Ethanol/30% Water/10% DMSO-50
.mu.L Stock 1, 10 .mu.L ethanol, 30 gL sterile water, 10 .mu.L
DMSO. 45,000 pmol/g .omega.-ACTX-Hv1a+2 50% Ethanol/30% Water/10%
DMSO-50 .mu.L Stock 1, 40 .mu.L sterile water, 10 .mu.L DMSO.
[0151] For Topical Assay using .omega.-ACTX-Hv1a+2 with MSO.RTM.
Surfactant:
5% MSO.RTM. (-ve control)-95 .mu.L Ethanol, 5 .mu.L MSO.RTM.
Concentrate. 1.25% MSO.RTM. (-ve control)-98.75 .mu.L Ethanol, 1.25
.mu.L MSO.RTM. Concentrate. 45,000 pmol/g .omega.-ACTX-Hv1a+2 5%
MSO.RTM.-50 .mu.L Stock 1, 45 .mu.L Ethanol, 5 .mu.L MSO
Concentrate. 45,000 pmol/g .omega.-ACTX-Hv1a+2 2.5% MSO.RTM.-25
.mu.L Stock 1, 23.75 gL Ethanol, 1.25 .mu.L MSO.RTM. Concentrate.
45,000 pmol/g .omega.-ACTX-Hv1a+2 1.25% MSO.RTM.-25 .mu.L Stock 1,
24.37 gL Ethanol, 0.625 gL MSO.RTM. Concentrate All treatment
mixtures were kept on ice for .about.1 hr. prior to administration
and application. Administration and application of the
formulation.
[0152] 2 .mu.L samples of each treatment mixture were spotted onto
the dorsal thorax of indivisual houseflies (10 houseflies treated
per mixture). A second group of ten flies were each treated with 2
.mu.L samples of the 45,000 pmol/g .omega.-ACTX-Hv1a+2 in 90%
Ethanol/10% DMSO but with the treatment applied to the ventral
abdominal surface of the flies rather than the dorsal thoracic
surface. After application, flies were kept in plastic containers
wells with ad libitum access to food (1:1 mixture of dry powdered
milk and table sugar) and water (presented in soaked cotton balls)
and observed every 8-24 hrs. for two days.
Results of Topical Application of .omega.-ACTX-Hv1a+2
[0153] Post-application mortality (and "twitching" and "moribund"
behavior, both presumably resulting from disruption of
physiological norms by action of the toxin) is summarized in Table
15, below.
TABLE-US-00026 TABLE 15 Results of topical assays of DMSO, ethanol
and MSO .RTM. preparations of .omega.-ACTX-Hv1a + 2, administered
topically. Dead Dead Dead Treatment N (4 hr) (14 hr) Dead (22 hr)
(38 hr) Dead (48 hr) -ve 90EtOH/ 10 0 0 0 0 0 10DMSO 45,000 pmol/g
10 1 2 3 5 5 90EtOH/ (1 twch) 10DMSO 45,0000 pmol/g 10 0 1 2 4 4
80EtOH/10H2O/ (1 twch) (1 twch) 10DMSO 45,0000 pmol/g 10 0 0 0 0 0
70EtOH/20H2O/ 10DMSO 45,0000 pmol/g 10 0 0 0 0 0 60EtOH/30H2O/
10DMSO 45,0000 pmol/g 10 0 0 0 1 1 50EtOH/40H2O/ 10DMSO 45,000
pmol/g 10 0 1 0 1 1 90H2O/10DMSO (3 twch) -ve 5% MSO .RTM. 10 0 0 0
0 1 -ve 1.25% 10 0 0 1 1 1 MSO .RTM. (1 morb) (1 morb) 45,000
pmol/g 10 2 4 4 4 5 5% MSO .RTM. (1 twch) (1 morb) (1 morb) (1
twch) 45,000 pmol/g 10 0 3 4 4 5 2.5% MSO .RTM. (1 twch) (1 twch)
45,000 pmol/g 10 1 1 1 5 6 1.25% MSO .RTM. (1 twch) (1 twch) 45,000
pmol/g 10 0 1 5 5 7 90EtOH/10DMSO (3 twch) (1 twch) (1 twch)
TUMMY
[0154] Number 1--addition of 10% water to precipitates of omega
toxin in ethanol/DMSO solutions appears to reduce topical
insecticidal activity of the precipitate under the conditions
tested above.
[0155] Number 2--addition of 20%, 30%, and 40% water to
precipitates of omega toxin in ethanol/DMSO solutions appears to
completely eliminate topical insecticidal activity of the
precipitate under the conditions tested above.
[0156] Number 3--solvation/dilution of toxin in 90% water/10%DMSO
results in a solution with no insecticidal activity under the
conditions tested above.
[0157] Number 4--replacement of 10% DMSO with either 1.25%
MSO.RTM., 2.5% MSO.RTM., or 5% MSO.RTM. apparently results in
mixtures with significant insecticidal activity under the
conditions tested above.
[0158] Number 5--under experimental conditions used above, ventral
abdominal application of the precipitate (of omega toxin in 90%
ethanol/10% DMSO) appears to induce insect mortality with speed and
effectiveness similar to, if not greater than, the induction of
mortality by dorsal thoracic application of the same precipitate
mixture. Since ventral abdominal application can be executed
roughly twice as quickly as dorsal thoracic application, this
points to a significant technical improvement for future topical
application bioassays.
[0159] Further examples of toxic peptides and the sequence
listing.
[0160] The toxic insect peptides refers to peptides that are not
from toxic insects, but that are toxic to insects. Their source
need not be insects. In the sequence listing of this application a
wide range of suitable toxic insect peptides are provided. This
small selection of about 174 peptides includes representative
peptides from spiders, scorpions and plants. Sequences 1-140 are
from funnel web spiders, sequences 141 to 171 are from scoprions
and sequences 172 to 174 are from plants. The sequence listing
includes peptide examples where the source is Oldenlandia affinis
which is known to produce a cyclic peptide referred to at a
"Kalata" type peptide. The Oldenlandia plant family is known to
produce peptides having insecticidal activity. Other insecticidal
peptides from plants are known. Numerous venomus spider peptides,
which are discussed throughout this application are also provided.
Sequence CWU 1
1
174141PRTAtrax robustus 1Gly Ser Gln Tyr Cys Ala Pro Ala Asp Gln
Pro Cys Ser Leu Asn Thr 1 5 10 15 Gln Pro Cys Cys Asp Asp Val Thr
Cys Thr Gln Glu Arg Asn Glu Asn 20 25 30 Gly His Thr Ala Tyr Tyr
Cys Arg Val 35 40 298PRTHadronyche versuta 2Met Lys Phe Ser Lys Leu
Ser Leu Thr Leu Ala Leu Ile Leu Thr Gln 1 5 10 15 Ala Leu Phe Val
Leu Cys Gly Lys Ile Asn Glu Asp Phe Met Lys Asn 20 25 30 Gly Leu
Glu Ser Gln Thr Leu His Asp Glu Ile Arg Lys Pro Ile Asp 35 40 45
Ser Glu Asn Pro Asp Thr Glu Arg Leu Leu Asp Cys Leu Leu Asp Asn 50
55 60 Arg Val Cys Ser Ser Asp Arg Asp Cys Cys Gly Met Thr Pro Ser
Cys 65 70 75 80 Thr Met Gly Leu Cys Val Pro Asn Val Gly Gly Leu Val
Gly Gly Ile 85 90 95 Leu Gly 398PRTAtrax robustus 3Met Lys Phe Ser
Lys Leu Ser Leu Thr Leu Ala Leu Ile Leu Thr Gln 1 5 10 15 Ala Leu
Phe Val Leu Cys Gly Lys Ile Asn Glu Asp Phe Met Lys Asn 20 25 30
Gly Leu Glu Ser Gln Thr Leu His Asp Glu Ile Arg Lys Pro Ile Asp 35
40 45 Ser Glu Asn Pro Asp Thr Glu Arg Leu Leu Asp Cys Leu Leu Asp
Asn 50 55 60 Arg Val Cys Ser Ser Asp Arg Asp Cys Cys Gly Met Thr
Pro Ser Cys 65 70 75 80 Thr Met Gly Leu Cys Val Pro Asn Val Gly Gly
Leu Val Gly Gly Ile 85 90 95 Leu Gly 498PRTHadronyche versuta 4Met
Lys Phe Ser Lys Leu Ser Leu Thr Leu Ala Leu Ile Leu Thr Gln 1 5 10
15 Ala Ile Phe Val Leu Cys Gly Lys Ile Asn Glu Asp Phe Met Lys Asn
20 25 30 Asp Leu Glu Ser Gln Ala Leu Arg Asp Glu Ile Arg Lys Pro
Ile Asp 35 40 45 Ser Glu Asn Pro Asp Thr Glu Arg Leu Leu Asp Cys
Leu Leu Asp Asn 50 55 60 Arg Val Cys Ser Ser Asp Lys Asp Cys Cys
Gly Met Thr Pro Ser Cys 65 70 75 80 Thr Met Gly Leu Cys Val Pro Ser
Val Gly Gly Leu Val Gly Gly Ile 85 90 95 Leu Gly 598PRTHadronyche
versuta 5Met Lys Phe Ser Lys Leu Ser Leu Thr Leu Ala Leu Ile Leu
Thr Gln 1 5 10 15 Ala Ile Phe Val Leu Cys Gly Lys Ile Asn Glu Asp
Phe Met Lys Asn 20 25 30 Asp Leu Glu Ser His Ala Leu His Asp Glu
Ile Arg Lys Pro Ile Asn 35 40 45 Ser Glu Asn Pro Asp Thr Glu Arg
Leu Leu Asp Cys Leu Leu Asp Asn 50 55 60 Arg Val Cys Ser Ser Asp
Lys Asp Cys Cys Gly Met Thr Pro Ser Cys 65 70 75 80 Thr Met Gly Leu
Cys Val Pro Ser Val Gly Gly Leu Val Gly Gly Ile 85 90 95 Leu Gly
698PRTHadronyche versuta 6Met Lys Phe Ser Lys Leu Ser Leu Thr Leu
Ala Leu Ile Leu Thr Gln 1 5 10 15 Ala Leu Phe Val Leu Cys Gly Lys
Ile Asn Glu Asp Phe Met Lys Asn 20 25 30 Gly Leu Glu Ser Gln Ala
Leu His Asp Glu Ile Arg Lys Pro Ile Asp 35 40 45 Ser Glu Asn Pro
Asp Thr Glu Arg Leu Leu Asp Cys Leu Leu Asp Asn 50 55 60 Arg Val
Cys Ser Ser Asp Lys Asp Cys Cys Gly Met Thr Pro Ser Cys 65 70 75 80
Thr Met Gly Leu Cys Val Pro Ser Val Gly Gly Leu Val Gly Gly Ile 85
90 95 Leu Gly 798PRTHadronyche versuta 7Met Lys Phe Ser Lys Leu Ser
Leu Thr Leu Ala Leu Ile Leu Thr Gln 1 5 10 15 Val Ile Phe Val Leu
Cys Gly Lys Ile Asn Glu Asp Phe Met Lys Asn 20 25 30 Gly Leu Glu
Ser Gln Ala Leu His Asp Glu Ile Arg Lys Pro Ile Asp 35 40 45 Ser
Glu Asn Pro Asp Thr Glu Arg Leu Leu Asp Cys Leu Leu Asp Asn 50 55
60 Arg Val Cys Ser Ser Asp Lys Asp Cys Cys Gly Met Thr Pro Ser Cys
65 70 75 80 Thr Met Gly Leu Cys Val Pro Ser Val Gly Gly Leu Val Gly
Gly Ile 85 90 95 Leu Gly 893PRTHadronyche versuta 8Met Lys Phe Ser
Lys Leu Ser Leu Thr Leu Ala Leu Ile Leu Thr Gln 1 5 10 15 Ala Leu
Phe Val Leu Cys Asp Phe Met Lys Asn Gly Leu Glu Ser Gln 20 25 30
Ala Leu His Asp Glu Ile Arg Lys Ser Ile Asp Ser Glu Asn Pro Asp 35
40 45 Thr Glu Arg Leu Leu Asp Cys Leu Leu Asp Asn Arg Val Cys Ser
Ser 50 55 60 Asp Lys Asp Cys Cys Gly Met Thr Pro Ser Cys Thr Met
Gly Leu Cys 65 70 75 80 Val Pro Ser Val Gly Gly Leu Val Gly Gly Ile
Leu Gly 85 90 998PRTAtrax robustus 9Met Lys Phe Ser Lys Leu Ser Leu
Thr Leu Ala Leu Ile Leu Thr Gln 1 5 10 15 Val Leu Phe Val Leu Cys
Gly Lys Ile Asn Glu Asp Phe Met Lys His 20 25 30 Gly Leu Glu Ser
Gln Ala Leu His Asp Glu Ile Arg Lys Pro Ile Asp 35 40 45 Ser Glu
Asn Pro Asp Thr Glu Arg Leu Leu Asp Cys Leu Leu Asp Asn 50 55 60
Arg Val Cys Ser Ser Asp Lys Asp Cys Cys Gly Met Thr Pro Ser Cys 65
70 75 80 Thr Met Gly Leu Cys Val Pro Ser Val Gly Gly Leu Val Gly
Gly Ile 85 90 95 Leu Gly 1098PRTHadronyche versuta 10Met Lys Phe
Ser Lys Leu Ser Leu Thr Leu Ala Leu Ile Leu Thr Gln 1 5 10 15 Ala
Ile Phe Val Leu Cys Gly Lys Ile Asn Glu Asp Phe Met Lys Asn 20 25
30 Asp Leu Glu Ser Gln Ala Leu His Asp Glu Ile Arg Lys Pro Ile Asn
35 40 45 Ser Glu Asn Pro Asp Thr Glu Arg Leu Leu Asp Cys Leu Leu
Asp Asn 50 55 60 Arg Val Cys Ser Ser Asp Lys Asp Cys Cys Gly Met
Thr Pro Ser Cys 65 70 75 80 Thr Met Gly Leu Cys Val Pro Ser Val Gly
Gly Leu Val Gly Gly Ile 85 90 95 Leu Gly 1198PRTHadronyche versuta
11Met Lys Phe Ser Lys Leu Ser Leu Thr Leu Ala Leu Ile Leu Thr Gln 1
5 10 15 Ala Leu Phe Val Leu Cys Gly Lys Ile Asn Glu Asp Phe Met Lys
Asn 20 25 30 Gly Leu Glu Ser Gln Ala Leu His Asp Glu Ile Arg Lys
Pro Ile Asp 35 40 45 Ser Glu Asn Pro Asp Thr Glu Arg Leu Leu Asp
Cys Leu Leu Asp Asn 50 55 60 Arg Val Cys Ser Ser Asp Arg Asp Cys
Cys Gly Met Thr Pro Ser Cys 65 70 75 80 Thr Met Gly Leu Cys Val Pro
Asn Val Gly Gly Leu Val Gly Asp Ile 85 90 95 Leu Gly
1297PRTHadronyche versuta 12Met Lys Phe Ser Lys Leu Ser Leu Thr Leu
Ala Leu Ile Leu Thr Gln 1 5 10 15 Val Leu Phe Val Leu Cys Gly Lys
Ile Glu Asp Phe Met Lys Asn Gly 20 25 30 Leu Glu Ser Gln Ala Leu
His Asp Glu Ile Arg Lys Pro Ile Asp Ser 35 40 45 Glu Asn Pro Asp
Thr Glu Arg Leu Leu Asp Cys Leu Leu Asp Asn Arg 50 55 60 Val Cys
Ser Ser Asp Lys Asp Cys Cys Gly Met Thr Pro Ser Cys Thr 65 70 75 80
Met Gly Leu Cys Val Pro Asn Val Gly Gly Leu Val Gly Gly Ile Leu 85
90 95 Gly 1393PRTHadronyche versuta 13Met Lys Phe Ser Lys Leu Ser
Leu Thr Leu Ala Leu Ile Leu Thr Gln 1 5 10 15 Ala Leu Phe Val Leu
Cys Asp Phe Met Lys Asn Gly Leu Glu Ser Gln 20 25 30 Ala Leu His
Asp Glu Ile Arg Lys Pro Ile Asp Ser Glu Asn Pro Asp 35 40 45 Thr
Glu Arg Leu Leu Asp Cys Leu Leu Asp Asn Arg Val Cys Ser Ser 50 55
60 Asp Lys Asp Cys Cys Gly Met Thr Pro Ser Cys Thr Met Gly Leu Cys
65 70 75 80 Val Pro Asn Val Gly Gly Leu Val Gly Gly Ile Leu Gly 85
90 1498PRTHadronyche versuta 14Met Lys Phe Ser Lys Leu Ser Leu Thr
Leu Ala Leu Ile Leu Thr Gln 1 5 10 15 Ala Leu Phe Val Leu Cys Gly
Lys Ile Asn Glu Asp Phe Met Lys Asn 20 25 30 Gly Leu Glu Ser Gln
Ala Leu His Asp Glu Ile Arg Lys Pro Ile Asp 35 40 45 Ser Glu Asn
Pro Asp Thr Glu Arg Leu Leu Asp Cys Leu Leu Asp Asn 50 55 60 Arg
Ile Cys Ser Ser Asp Lys Asp Cys Cys Gly Met Thr Pro Ser Cys 65 70
75 80 Thr Met Gly Leu Cys Val Pro Asn Val Gly Gly Leu Val Gly Gly
Ile 85 90 95 Leu Gly 1598PRTHadronyche versuta 15Met Lys Phe Ser
Lys Leu Ser Leu Thr Leu Ala Leu Ile Leu Thr Gln 1 5 10 15 Ala Leu
Phe Val Leu Cys Gly Lys Ile Asn Glu Asp Phe Met Lys Asn 20 25 30
Gly Leu Glu Ser Gln Ala Leu His Asp Glu Ile Arg Lys Pro Ile Asp 35
40 45 Ser Glu Asn Pro Asp Thr Glu Arg Leu Leu Asp Cys Leu Leu Asp
Asn 50 55 60 Arg Val Cys Ser Ser Asp Lys Asp Cys Cys Gly Met Thr
Pro Ser Cys 65 70 75 80 Thr Met Gly Leu Cys Val Pro Asn Val Gly Gly
Leu Val Gly Gly Ile 85 90 95 Leu Gly 1698PRTHadronyche versuta
16Met Lys Phe Ser Lys Leu Ser Leu Thr Leu Ala Leu Ile Leu Thr Gln 1
5 10 15 Ala Ile Phe Val Leu Cys Gly Lys Ile Asn Glu Asp Phe Met Lys
Asn 20 25 30 Asp Leu Glu Ser Gln Ala Leu His Asp Glu Ile Arg Lys
Pro Ile Asn 35 40 45 Ser Glu Asn Pro Asp Thr Glu Arg Leu Leu Asp
Cys Leu Leu Asp Ser 50 55 60 Arg Val Cys Ser Ser Asp Lys Asp Cys
Cys Gly Met Thr Pro Ser Cys 65 70 75 80 Thr Met Gly Leu Cys Val Pro
Ser Val Gly Gly Leu Val Gly Gly Ile 85 90 95 Leu Gly 1796PRTAtrax
robustus 17Met Lys Phe Ser Lys Leu Ser Ile Thr Leu Ala Val Ile Leu
Thr Gln 1 5 10 15 Ala Val Phe Val Phe Cys Gly Met Thr Asn Glu Asp
Phe Met Glu Lys 20 25 30 Gly Leu Glu Ser Asn Glu Leu Pro Asp Ala
Ile Lys Lys Pro Val Asn 35 40 45 Ser Gly Lys Pro Asp Thr Lys Arg
Leu Leu Asp Cys Val Leu Ser Arg 50 55 60 Met Cys Phe Ser Asn Ala
Asn Cys Cys Gly Leu Thr Pro Pro Cys Lys 65 70 75 80 Met Gly Leu Cys
Val Pro Asn Val Gly Gly Leu Leu Gly Gly Ile Leu 85 90 95
1896PRTAtrax robustus 18Met Lys Phe Ser Lys Leu Ser Ile Thr Leu Ala
Val Ile Leu Thr Gln 1 5 10 15 Ala Val Phe Val Phe Cys Gly Met Thr
Asn Glu Asp Phe Met Glu Lys 20 25 30 Gly Leu Glu Ser Asn Glu Leu
His Asp Ala Ile Lys Lys Pro Val Asn 35 40 45 Ser Gly Lys Pro Asp
Thr Glu Arg Leu Leu Asp Cys Val Leu Ser Arg 50 55 60 Met Cys Ser
Ser Asp Ala Asn Cys Cys Gly Leu Thr Pro Thr Cys Lys 65 70 75 80 Met
Gly Leu Cys Val Pro Asn Val Gly Gly Leu Leu Gly Gly Ile Leu 85 90
95 19101PRTHadronyche versuta 19Met Lys Phe Ser Lys Leu Ser Leu Thr
Leu Ala Leu Ile Leu Thr Gln 1 5 10 15 Ala Leu Phe Val Leu Cys Gly
Lys Ile Asn Glu Asp Phe Met Glu His 20 25 30 Gly Leu Glu Ser His
Ala Leu His Asp Glu Ile Arg Lys Pro Ile Asp 35 40 45 Thr Glu Lys
Ala Asp Ala Glu Arg Leu Val Asp Cys Val Val Asn Thr 50 55 60 Leu
Gly Cys Ser Ser Asp Lys Asp Cys Cys Gly Met Thr Pro Ser Cys 65 70
75 80 Thr Leu Gly Ile Cys Ala Pro Ser Val Arg Gly Leu Val Gly Gly
Leu 85 90 95 Leu Gly Arg Ala Leu 100 20101PRTHadronyche versuta
20Met Lys Phe Ser Lys Leu Ser Leu Thr Leu Ala Leu Ile Leu Thr Gln 1
5 10 15 Ala Leu Phe Val Leu Cys Met Lys Ile Asn Glu Asp Phe Met Glu
Asn 20 25 30 Gly Leu Glu Ser His Ala Leu His Asp Glu Ile Arg Lys
Pro Ile Asp 35 40 45 Thr Glu Lys Ala Asp Ala Glu Arg Leu Val Asp
Cys Val Val Asn Thr 50 55 60 Leu Gly Cys Ser Ser Asp Lys Asp Cys
Cys Gly Met Thr Pro Ser Cys 65 70 75 80 Thr Leu Gly Ile Cys Ala Pro
Ser Val Gly Gly Leu Val Gly Gly Leu 85 90 95 Leu Gly Arg Ala Leu
100 21101PRTHadronyche versuta 21Met Lys Phe Ser Lys Leu Ser Leu
Thr Phe Ala Leu Ile Leu Thr Gln 1 5 10 15 Ala Leu Phe Val Leu Cys
Gly Lys Ile Asn Glu Asp Phe Met Asp Asn 20 25 30 Gly Leu Glu Ser
His Ala Leu His Asp Glu Ile Arg Lys Pro Ile His 35 40 45 Thr Glu
Lys Ala Asp Ala Glu Arg Leu Val Asp Cys Val Leu Asn Thr 50 55 60
Leu Gly Cys Ser Ser Asp Lys Asp Cys Cys Gly Met Thr Pro Ser Cys 65
70 75 80 Thr Leu Gly Ile Cys Ala Pro Ser Val Gly Gly Leu Val Gly
Gly Leu 85 90 95 Leu Gly Arg Ala Leu 100 22101PRTHadronyche infensa
22Met Lys Phe Ser Lys Leu Ser Leu Thr Leu Ala Leu Ile Leu Thr Gln 1
5 10 15 Ala Leu Phe Val Leu Cys Gly Lys Ile Asn Glu Asp Phe Met Glu
His 20 25 30 Gly Leu Glu Ser His Ala Leu His Asp Glu Ile Arg Lys
Pro Ile Asp 35 40 45 Thr Glu Lys Ala Asp Ala Glu Arg Leu Val Asp
Cys Val Val Asn Thr 50 55 60 Leu Gly Cys Ser Ser Asp Lys Asp Cys
Cys Gly Met Thr Pro Ser Cys 65 70 75 80 Thr Leu Gly Ile Cys Ala Pro
Ser Val Gly Gly Leu Val Gly Gly Leu 85 90 95 Leu Gly Arg Ala Leu
100 23101PRTHadronyche infensa 23Met Lys Phe Ser Lys Leu Ser Val
Thr Leu Ala Leu Ile Leu Thr Gln 1 5 10 15 Thr Leu Leu Val Leu Cys
Gly Lys Ile Asn Glu Asp Phe Met Glu Asn 20 25 30 Gly Leu Glu Ser
His Ala Leu His Asp Glu Ile Arg Lys Pro Ile Asp 35 40 45 Thr Asp
Lys Ala Tyr Ala Glu Arg Val Leu Asp Cys Val Val Asn Thr 50 55 60
Leu Gly Cys Ser Ser Asp Lys Asp Cys Cys Gly Met Thr Pro Ser Cys 65
70 75 80 Thr Leu Gly Ile Cys Ala Pro Ser Val Gly Gly Leu Val Gly
Gly Leu 85 90 95 Leu Gly Arg Ala Leu 100 24101PRTHadronyche versuta
24Met Lys Phe Ser Lys Leu Ser Leu Thr Leu Ala Leu Ile Leu Thr Gln 1
5 10 15 Ala Leu Phe Val Leu Cys Gly Lys Ile Asn Glu Asp Phe Met Glu
Asn 20
25 30 Gly Leu Glu Ser His Ala Leu His Asp Glu Ile Arg Lys Pro Ile
Asp 35 40 45 Thr Glu Lys Ala Asp Ala Glu Arg Leu Val Asp Cys Val
Val Asn Thr 50 55 60 Leu Gly Cys Ser Ser Asp Lys Asp Cys Cys Gly
Met Thr Pro Ser Cys 65 70 75 80 Thr Leu Gly Ile Cys Ala Pro Ser Val
Gly Gly Leu Val Gly Gly Leu 85 90 95 Leu Gly Arg Ala Leu 100
25101PRTHadronyche versuta 25Met Lys Phe Ser Lys Leu Ser Leu Thr
Leu Ala Leu Ile Leu Thr Gln 1 5 10 15 Ala Leu Phe Val Leu Cys Gly
Lys Ile Asn Glu Asp Phe Met Glu Asn 20 25 30 Gly Leu Glu Ser His
Ala Leu His Asp Glu Ile Arg Lys Pro Ile Asp 35 40 45 Thr Glu Lys
Ala Asp Ala Glu Arg Val Leu Asp Cys Val Val Asn Thr 50 55 60 Leu
Gly Cys Ser Ser Asp Lys Asp Cys Cys Gly Met Thr Pro Ser Cys 65 70
75 80 Thr Leu Gly Ile Cys Ala Pro Ser Val Gly Gly Leu Val Gly Gly
Leu 85 90 95 Leu Gly Arg Ala Leu 100 26100PRTHadronyche versuta
26Met Lys Phe Ser Lys Leu Ser Leu Thr Leu Ala Leu Ile Leu Thr Gln 1
5 10 15 Ala Leu Phe Val Leu Cys Gly Lys Ile Asn Glu Asp Phe Met Glu
Asn 20 25 30 Gly Leu Glu Ser His Ala Leu His Asp Glu Ile Arg Lys
Pro Ile Asp 35 40 45 Thr Glu Lys Ala Asp Ala Glu Arg Leu Val Asp
Cys Val Val Asn Thr 50 55 60 Leu Gly Cys Ser Ser Asp Lys Asp Cys
Cys Gly Met Thr Pro Ser Cys 65 70 75 80 Thr Leu Gly Ile Cys Ala Pro
Ser Val Gly Leu Val Gly Gly Leu Leu 85 90 95 Gly Arg Ala Leu 100
2797PRTAtrax robustus 27Met Lys Phe Ser Lys Leu Ser Ile Thr Leu Ala
Val Ile Leu Thr Gln 1 5 10 15 Ala Val Phe Val Phe Cys Gly Met Thr
Asn Glu Asp Phe Met Glu Lys 20 25 30 Gly Phe Lys Ser Asn Asp Leu
Gln Tyr Ala Ile Lys Gln Pro Val Asn 35 40 45 Ser Gly Lys Pro Asp
Thr Glu Arg Leu Leu Asp Cys Val Leu Ser Arg 50 55 60 Val Cys Ser
Ser Asp Glu Asn Cys Cys Gly Leu Thr Pro Thr Cys Thr 65 70 75 80 Met
Gly Leu Cys Val Pro Asn Val Gly Gly Leu Leu Gly Gly Leu Leu 85 90
95 Ser 2897PRTAtrax robustus 28Met Lys Phe Ser Lys Leu Ser Ile Thr
Leu Val Val Ile Leu Thr Gln 1 5 10 15 Ala Val Phe Val Phe Cys Gly
Met Thr Asn Glu Asp Phe Met Glu Lys 20 25 30 Gly Phe Lys Ser Asn
Asp Leu Gln Tyr Ala Ile Arg Gln Pro Val Asn 35 40 45 Ser Gly Lys
Pro Asp Thr Glu Arg Leu Leu Asp Cys Val Leu Ser Arg 50 55 60 Val
Cys Ser Ser Asp Glu Asn Cys Cys Gly Leu Thr Pro Thr Cys Thr 65 70
75 80 Met Gly Leu Cys Val Pro Asn Val Gly Gly Leu Leu Gly Gly Leu
Leu 85 90 95 Ser 29101PRTHadronyche versuta 29Met Lys Phe Ser Lys
Leu Ser Leu Thr Leu Ala Leu Ile Leu Thr Gln 1 5 10 15 Ala Leu Leu
Val Leu Cys Gly Lys Ile Asn Glu Asp Phe Met Glu Asn 20 25 30 Gly
Leu Glu Ser His Ala Leu His Asp Glu Ile Arg Lys Pro Leu Asp 35 40
45 Thr Glu Asn Pro Asp Thr Glu Arg Gln Leu Asp Cys Val Leu Asn Thr
50 55 60 Leu Gly Cys Ser Ser Asp Lys Asp Cys Cys Gly Met Thr Pro
Ser Cys 65 70 75 80 Thr Leu Gly Ile Cys Ala Pro Asn Val Gly Gly Leu
Val Gly Gly Leu 85 90 95 Leu Gly Arg Ala Leu 100 30101PRTHadronyche
versuta 30Met Lys Phe Ser Lys Leu Ser Leu Thr Leu Ala Leu Ile Leu
Thr Gln 1 5 10 15 Val Leu Leu Val Val Cys Gly Lys Ile Asn Glu Asp
Phe Met Glu Asn 20 25 30 Gly Leu Glu Ser His Ala Leu His Asp Glu
Ile Arg Lys Pro Ile Asp 35 40 45 Thr Glu Lys Ala Asp Ala Glu Arg
Val Leu Asp Cys Val Val Asn Thr 50 55 60 Leu Gly Cys Ser Ser Asp
Lys Asp Cys Cys Gly Met Thr Pro Ser Cys 65 70 75 80 Thr Leu Gly Ile
Cys Ala Pro Ser Val Gly Gly Ile Val Gly Gly Leu 85 90 95 Leu Gly
Arg Ala Leu 100 31101PRTHadronyche versuta 31Met Lys Phe Ser Lys
Leu Ser Leu Thr Leu Ala Leu Ile Leu Ala Gln 1 5 10 15 Ala Ile Phe
Val Leu Cys Gly Lys Ile Asn Glu Asp Phe Met Glu Asn 20 25 30 Gly
Leu Glu Ser His Ala Leu His Asp Glu Ile Arg Lys Pro Ile Asp 35 40
45 Thr Glu Lys Ala Asp Ala Glu Arg Val Val Asp Cys Val Leu Asn Thr
50 55 60 Leu Gly Cys Ser Ser Asp Lys Asp Cys Cys Gly Met Thr Pro
Ser Cys 65 70 75 80 Thr Leu Gly Ile Cys Ala Pro Ser Val Gly Gly Leu
Val Gly Gly Leu 85 90 95 Leu Gly Arg Ala Leu 100 32101PRTHadronyche
versuta 32Met Lys Phe Ser Lys Leu Ser Leu Thr Leu Ala Leu Ile Leu
Thr Gln 1 5 10 15 Ala Leu Leu Val Val Cys Gly Lys Ile Asn Glu Asp
Phe Met Glu Asn 20 25 30 Gly Leu Glu Ser His Ala Leu His Asp Glu
Ile Arg Lys Pro Ile Asp 35 40 45 Thr Glu Lys Ala Asp Ala Glu Arg
Val Leu Asp Cys Val Val Asn Ile 50 55 60 Leu Gly Cys Ser Ser Asp
Lys Asp Cys Cys Gly Met Thr Pro Ser Cys 65 70 75 80 Thr Leu Gly Ile
Cys Ala Pro Ser Val Gly Gly Ile Val Gly Gly Leu 85 90 95 Leu Gly
Arg Ala Leu 100 33101PRTHadronyche versuta 33Met Lys Phe Ser Lys
Leu Ser Leu Thr Leu Ala Leu Ile Leu Thr Gln 1 5 10 15 Ala Leu Leu
Val Val Cys Gly Lys Ile Asn Glu Asp Phe Met Glu Asn 20 25 30 Gly
Leu Glu Ser His Ala Leu His Asp Glu Ile Arg Lys Pro Ile Asp 35 40
45 Thr Glu Lys Ala Asp Ala Glu Arg Val Leu Asp Cys Val Val Asn Thr
50 55 60 Leu Gly Cys Ser Ser Asp Lys Asp Cys Cys Gly Met Thr Pro
Ser Cys 65 70 75 80 Thr Leu Gly Ile Cys Ala Pro Ser Val Gly Gly Ile
Val Gly Gly Leu 85 90 95 Leu Gly Arg Ala Leu 100 34101PRTHadronyche
infensa 34Met Lys Phe Ser Lys Leu Ser Leu Thr Leu Ala Leu Ile Leu
Thr Gln 1 5 10 15 Ala Leu Leu Val Val Cys Gly Lys Ile Asn Glu Asp
Phe Met Glu Asn 20 25 30 Gly Leu Glu Ser His Ala Leu His Asp Glu
Ile Arg Lys Ser Ile Asp 35 40 45 Thr Glu Lys Ala Asp Ala Glu Arg
Val Leu Asp Cys Val Val Asn Thr 50 55 60 Leu Gly Cys Ser Ser Asp
Lys Asp Cys Cys Gly Met Thr Pro Ser Cys 65 70 75 80 Thr Leu Gly Ile
Cys Ala Pro Ser Val Gly Gly Ile Val Gly Gly Leu 85 90 95 Leu Gly
Arg Ala Leu 100 3595PRTHadronyche versuta 35Met Lys Phe Ser Lys Leu
Ser Leu Thr Phe Ala Leu Ile Leu Thr Gln 1 5 10 15 Thr Leu Leu Val
Leu Cys Asp Phe Met Glu Asn Gly Leu Glu Ser His 20 25 30 Ala Leu
His Asp Glu Ile Arg Lys Pro Ile Asp Thr Glu Lys Ala Asp 35 40 45
Ala Glu Arg Val Leu Asp Cys Val Val Asn Thr Leu Gly Cys Ser Ser 50
55 60 Asp Lys Asp Cys Cys Gly Met Thr Pro Ser Cys Thr Leu Gly Ile
Cys 65 70 75 80 Ala Pro Ser Val Gly Gly Leu Val Gly Gly Leu Leu Gly
Arg Ala 85 90 95 3676PRTHadronyche versuta 36Met Asn Thr Ala Thr
Gly Phe Ile Val Leu Leu Val Leu Ala Thr Val 1 5 10 15 Leu Gly Gly
Val Glu Ala Gly Glu Ser His Met Arg Lys Asp Ala Met 20 25 30 Gly
Arg Val Arg Arg Gln Tyr Cys Val Pro Val Asp Gln Pro Cys Ser 35 40
45 Leu Asn Thr Gln Pro Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Arg
50 55 60 Asn Glu Asn Gly His Thr Val Tyr Tyr Cys Arg Ala 65 70 75
3739PRTHadronyche versuta 37Gln Tyr Cys Val Pro Val Asp Gln Pro Cys
Ser Leu Asn Thr Gln Pro 1 5 10 15 Cys Cys Asp Asp Ala Thr Cys Thr
Gln Glu Arg Asn Glu Asn Gly His 20 25 30 Thr Val Tyr Tyr Cys Arg
Ala 35 3839PRTHadronyche versuta 38Gly Ser Cys Val Pro Val Asp Gln
Pro Cys Ser Leu Asn Thr Gln Pro 1 5 10 15 Cys Cys Asp Asp Ala Thr
Cys Thr Gln Glu Arg Asn Glu Asn Gly His 20 25 30 Thr Val Tyr Tyr
Cys Arg Ala 35 3975PRTHadronyche versuta 39Met Asn Thr Ala Thr Gly
Phe Ile Val Leu Leu Val Leu Ala Thr Ile 1 5 10 15 Leu Gly Gly Ile
Glu Ala Gly Glu Ser His Met Arg Lys Asp Ala Met 20 25 30 Gly Arg
Val Arg Arg Gln Tyr Cys Val Pro Val Asp Gln Pro Cys Ser 35 40 45
Leu Asn Thr Gln Pro Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Arg 50
55 60 Asn Glu Asn Gly His Thr Val Tyr Tyr Cys Arg 65 70 75
4038PRTHadronyche versuta 40Gln Tyr Cys Val Pro Val Asp Gln Pro Cys
Ser Leu Asn Thr Gln Pro 1 5 10 15 Cys Cys Asp Asp Ala Thr Cys Thr
Gln Glu Arg Asn Glu Asn Gly His 20 25 30 Thr Val Tyr Tyr Cys Arg 35
4175PRTHadronyche versuta 41Met Asn Thr Ala Thr Gly Phe Ile Val Leu
Leu Val Leu Ala Thr Val 1 5 10 15 Leu Gly Gly Ile Glu Ala Gly Glu
Ser His Met Arg Lys Asp Ala Met 20 25 30 Gly Arg Val Arg Arg Gln
Tyr Cys Val Pro Val Asp Gln Pro Cys Ser 35 40 45 Leu Asn Thr Gln
Pro Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Leu 50 55 60 Asn Glu
Asn Asp Asn Thr Val Tyr Tyr Cys Arg 65 70 75 4238PRTHadronyche
versuta 42Gln Tyr Cys Val Pro Val Asp Gln Pro Cys Ser Leu Asn Thr
Gln Pro 1 5 10 15 Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Leu Asn
Glu Asn Asp Asn 20 25 30 Thr Val Tyr Tyr Cys Arg 35
4376PRTHadronyche versuta 43Met Asn Thr Ala Thr Gly Phe Ile Val Leu
Leu Val Leu Ala Thr Ile 1 5 10 15 Leu Gly Gly Ile Glu Ala Gly Glu
Ser His Met Arg Lys Asp Ala Met 20 25 30 Gly Arg Val Arg Arg Gln
Tyr Cys Val Pro Val Asp Gln Pro Cys Ser 35 40 45 Leu Asn Thr Gln
Pro Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Arg 50 55 60 Asn Glu
Asn Gly His Thr Val Tyr Tyr Cys Arg Ala 65 70 75 4439PRTHadronyche
versuta 44Gln Tyr Cys Val Pro Val Asp Gln Pro Cys Ser Leu Asn Thr
Gln Pro 1 5 10 15 Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Arg Asn
Glu Asn Gly His 20 25 30 Thr Val Tyr Tyr Cys Arg Ala 35
4576PRTHadronyche versuta 45Met Asn Thr Ala Thr Gly Phe Ile Val Leu
Leu Val Leu Ala Thr Val 1 5 10 15 Leu Gly Gly Ile Glu Ala Gly Glu
Ser His Met Arg Lys Asp Ala Met 20 25 30 Gly Arg Val Arg Arg Gln
Tyr Cys Val Pro Val Asp Gln Pro Cys Ser 35 40 45 Leu Asn Thr Gln
Pro Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Leu 50 55 60 Asn Glu
Asn Ala Asn Pro Val Tyr Tyr Cys Arg Ala 65 70 75 4639PRTHadronyche
versuta 46Gln Tyr Cys Val Pro Val Asp Gln Pro Cys Ser Leu Asn Thr
Gln Pro 1 5 10 15 Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Leu Asn
Glu Asn Ala Asn 20 25 30 Pro Val Tyr Tyr Cys Arg Ala 35
4776PRTHadronyche versuta 47Met Asn Thr Thr Thr Gly Phe Ile Val Leu
Leu Val Leu Ala Thr Ile 1 5 10 15 Leu Gly Gly Ile Glu Ala Gly Glu
Ser His Met Arg Lys Asp Ala Met 20 25 30 Gly Arg Val Arg Arg Gln
Tyr Cys Val Pro Val Asp Gln Pro Cys Ser 35 40 45 Leu Asn Thr Gln
Pro Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Leu 50 55 60 Asn Glu
Asn Asp Asn Thr Val Tyr Tyr Cys Arg Ala 65 70 75 4839PRTHadronyche
versuta 48Gln Tyr Cys Val Pro Val Asp Gln Pro Cys Ser Leu Asn Thr
Gln Pro 1 5 10 15 Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Leu Asn
Glu Asn Asp Asn 20 25 30 Thr Val Tyr Tyr Cys Arg Ala 35
4976PRTHadronyche versuta 49Met Asn Thr Ala Thr Gly Phe Ile Val Leu
Leu Val Leu Ala Thr Val 1 5 10 15 Leu Gly Gly Ile Glu Ala Gly Glu
Ser His Met Arg Lys Asp Ala Met 20 25 30 Gly Arg Val Arg Arg Gln
Tyr Cys Val Pro Val Asp Gln Pro Cys Ser 35 40 45 Leu Asn Thr Gln
Pro Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Leu 50 55 60 Asn Glu
Asn Asp Asn Thr Val Tyr Tyr Cys Arg Ala 65 70 75 5039PRTHadronyche
versuta 50Gln Tyr Cys Val Pro Val Asp Gln Pro Cys Ser Leu Asn Thr
Gln Pro 1 5 10 15 Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Leu Asn
Glu Asn Asp Asn 20 25 30 Thr Val Tyr Tyr Cys Arg Ala 35
5176PRTHadronyche versuta 51Met Asn Thr Ala Thr Gly Phe Ile Val Phe
Leu Val Leu Ala Thr Val 1 5 10 15 Leu Gly Gly Ile Glu Ala Gly Glu
Ser His Met Arg Lys Asp Ala Met 20 25 30 Gly Arg Val Arg Arg Gln
Tyr Cys Val Pro Val Asp Gln Pro Cys Ser 35 40 45 Leu Asn Thr Gln
Pro Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Leu 50 55 60 Asn Glu
Asn Asp Asn Thr Val Tyr Tyr Cys Arg Ala 65 70 75 5239PRTHadronyche
versuta 52Gln Tyr Cys Val Pro Val Asp Gln Pro Cys Ser Leu Asn Thr
Gln Pro 1 5 10 15 Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Leu Asn
Glu Asn Asp Asn 20 25 30 Thr Val Tyr Tyr Cys Arg Ala 35
5376PRTHadronyche versuta 53Met Asn Thr Ala Thr Gly Phe Ile Val Leu
Leu Val Leu Ala Thr Val 1 5 10 15 Leu Gly Gly Ile Glu Ala Arg Glu
Ser His Met Arg Lys Asp Ala Met 20 25 30 Gly Arg Val Arg Arg Gln
Tyr Cys Val Pro Val Asp Gln Pro Cys Ser 35 40 45 Leu Asn Thr Gln
Pro Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Leu 50 55 60 Asn Glu
Asn Asp Asn Thr
Val Tyr Tyr Cys Arg Ala 65 70 75 5439PRTHadronyche versuta 54Gln
Tyr Cys Val Pro Val Asp Gln Pro Cys Ser Leu Asn Thr Gln Pro 1 5 10
15 Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Leu Asn Glu Asn Asp Asn
20 25 30 Thr Val Tyr Tyr Cys Arg Ala 35 5537PRTHadronyche versuta
55Ser Pro Thr Cys Ile Pro Ser Gly Gln Pro Cys Pro Tyr Asn Glu Asn 1
5 10 15 Cys Cys Ser Gln Ser Cys Thr Phe Lys Glu Asn Glu Asn Gly Asn
Thr 20 25 30 Val Lys Arg Cys Asp 35 5645PRTHadronyche versuta 56Leu
Leu Ala Cys Leu Phe Gly Asn Gly Arg Cys Ser Ser Asn Arg Asp 1 5 10
15 Cys Cys Glu Leu Thr Pro Val Cys Lys Arg Gly Ser Cys Val Ser Ser
20 25 30 Gly Pro Gly Leu Val Gly Gly Ile Leu Gly Gly Ile Leu 35 40
45 5719PRTHadronyche versuta 57Glu Asp Thr Arg Ala Asp Leu Gln Gly
Gly Glu Ala Ala Glu Lys Val 1 5 10 15 Phe Arg Arg 5815PRTHadronyche
versuta 58Gly Glu Ser His Val Arg Glu Asp Ala Met Gly Arg Ala Arg
Arg 1 5 10 15 5978PRTAtrax robustus 59Met Asn Thr Ala Thr Gly Val
Ile Ala Leu Leu Val Leu Ala Thr Val 1 5 10 15 Ile Gly Cys Ile Glu
Ala Glu Asp Thr Arg Ala Asp Leu Gln Gly Gly 20 25 30 Glu Ala Ala
Glu Lys Val Phe Arg Arg Ser Pro Thr Cys Ile Pro Ser 35 40 45 Gly
Gln Pro Cys Pro Tyr Asn Glu Asn Cys Cys Ser Gln Ser Cys Thr 50 55
60 Phe Lys Glu Asn Glu Asn Gly Asn Thr Val Lys Arg Cys Asp 65 70 75
6037PRTAtrax robustus 60Ser Pro Thr Cys Ile Pro Ser Gly Gln Pro Cys
Pro Tyr Asn Glu Asn 1 5 10 15 Cys Cys Ser Gln Ser Cys Thr Phe Lys
Glu Asn Glu Asn Gly Asn Thr 20 25 30 Val Lys Arg Cys Asp 35
6178PRTAtrax robustus 61Met Asn Thr Ala Thr Gly Val Ile Ala Leu Leu
Val Leu Val Thr Val 1 5 10 15 Ile Gly Cys Ile Glu Ala Glu Asp Thr
Arg Ala Asp Leu Gln Gly Gly 20 25 30 Glu Ala Ala Glu Lys Val Phe
Arg Arg Ser Pro Thr Cys Ile Pro Ser 35 40 45 Gly Gln Pro Cys Pro
Tyr Asn Glu Asn Cys Cys Ser Gln Ser Cys Thr 50 55 60 Phe Lys Glu
Asn Glu Asn Gly Asn Thr Val Lys Arg Cys Asp 65 70 75 6237PRTAtrax
robustus 62Ser Pro Thr Cys Ile Pro Ser Gly Gln Pro Cys Pro Tyr Asn
Glu Asn 1 5 10 15 Cys Cys Ser Gln Ser Cys Thr Phe Lys Glu Asn Glu
Asn Gly Asn Thr 20 25 30 Val Lys Arg Cys Asp 35 6378PRTAtrax
robustus 63Met Asn Thr Ala Thr Gly Val Ile Ala Leu Leu Val Leu Ala
Thr Val 1 5 10 15 Ile Gly Cys Ile Glu Ala Glu Asp Thr Arg Ala Asp
Leu Gln Gly Gly 20 25 30 Glu Ala Ala Glu Lys Val Phe Arg Arg Ser
Pro Thr Cys Ile Pro Ser 35 40 45 Gly Gln Pro Cys Pro Tyr Asn Glu
Asn Cys Cys Ser Gln Ser Cys Thr 50 55 60 Phe Lys Glu Asn Glu Thr
Gly Asn Thr Val Lys Arg Cys Asp 65 70 75 6437PRTAtrax robustus
64Ser Pro Thr Cys Ile Pro Ser Gly Gln Pro Cys Pro Tyr Asn Glu Asn 1
5 10 15 Cys Cys Ser Gln Ser Cys Thr Phe Lys Glu Asn Glu Thr Gly Asn
Thr 20 25 30 Val Lys Arg Cys Asp 35 6578PRTAtrax robustus 65Met Asn
Thr Ala Thr Gly Val Ile Ala Leu Leu Val Leu Ala Thr Val 1 5 10 15
Ile Gly Cys Ile Glu Ala Glu Asp Thr Arg Ala Asp Leu Gln Gly Gly 20
25 30 Glu Ala Ala Glu Lys Val Phe Arg Arg Ser Pro Thr Cys Ile Pro
Ser 35 40 45 Gly Gln Pro Cys Pro Tyr Asn Glu Asn Cys Cys Ser Gln
Ser Cys Thr 50 55 60 Phe Lys Glu Asn Glu Asn Ala Asn Thr Val Lys
Arg Cys Asp 65 70 75 6637PRTAtrax robustus 66Ser Pro Thr Cys Ile
Pro Ser Gly Gln Pro Cys Pro Tyr Asn Glu Asn 1 5 10 15 Cys Cys Ser
Gln Ser Cys Thr Phe Lys Glu Asn Glu Asn Ala Asn Thr 20 25 30 Val
Lys Arg Cys Asp 35 6778PRTAtrax robustus 67Met Asn Thr Ala Thr Gly
Val Ile Ala Leu Leu Val Leu Ala Thr Val 1 5 10 15 Ile Gly Cys Ile
Glu Ala Glu Asp Thr Arg Ala Asp Leu Gln Gly Gly 20 25 30 Glu Ala
Ala Glu Lys Val Phe Arg Arg Ser Pro Thr Cys Ile Pro Ser 35 40 45
Gly Gln Pro Cys Pro Tyr Asn Glu Asn Cys Cys Ser Lys Ser Cys Thr 50
55 60 Tyr Lys Glu Asn Glu Asn Gly Asn Thr Val Gln Arg Cys Asp 65 70
75 6837PRTAtrax robustus 68Ser Pro Thr Cys Ile Pro Ser Gly Gln Pro
Cys Pro Tyr Asn Glu Asn 1 5 10 15 Cys Cys Ser Lys Ser Cys Thr Tyr
Lys Glu Asn Glu Asn Gly Asn Thr 20 25 30 Val Gln Arg Cys Asp 35
6973PRTAtrax robustus 69Met Asn Thr Ala Thr Gly Phe Ile Val Leu Leu
Val Leu Ala Thr Val 1 5 10 15 Leu Gly Cys Ile Glu Ala Gly Glu Ser
His Val Arg Glu Asp Ala Met 20 25 30 Gly Arg Ala Arg Arg Gly Ala
Cys Thr Pro Thr Gly Gln Pro Cys Pro 35 40 45 Tyr Asn Glu Ser Cys
Cys Ser Gly Ser Cys Gln Glu Gln Leu Asn Glu 50 55 60 Asn Gly His
Thr Val Lys Arg Cys Val 65 70 7036PRTAtrax robustus 70Gly Ala Cys
Thr Pro Thr Gly Gln Pro Cys Pro Tyr Asn Glu Ser Cys 1 5 10 15 Cys
Ser Gly Ser Cys Gln Glu Gln Leu Asn Glu Asn Gly His Thr Val 20 25
30 Lys Arg Cys Val 35 7179PRTHadronyche versuta 71Met Asn Thr Ala
Thr Gly Phe Ile Val Leu Leu Val Leu Ala Thr Val 1 5 10 15 Ile Gly
Cys Ile Ser Ala Asp Phe Gln Gly Gly Phe Glu Pro Tyr Glu 20 25 30
Gly Glu Asp Ala Glu Arg Ile Phe Arg Arg Ser Pro Thr Cys Ile Pro 35
40 45 Thr Gly Gln Pro Cys Pro Tyr Asn Glu Asn Cys Cys Ser Gln Ser
Cys 50 55 60 Thr Tyr Lys Ala Asn Glu Asn Gly Asn Gln Val Lys Gly
Cys Asp 65 70 75 7237PRTHadronyche versuta 72Ser Pro Thr Cys Ile
Pro Thr Gly Gln Pro Cys Pro Tyr Asn Glu Asn 1 5 10 15 Cys Cys Ser
Gln Ser Cys Thr Tyr Lys Ala Asn Glu Asn Gly Asn Gln 20 25 30 Val
Lys Gly Cys Asp 35 7379PRTHadronyche versuta 73Met Asn Thr Ala Thr
Gly Phe Ile Val Leu Leu Val Leu Ala Thr Val 1 5 10 15 Ile Gly Cys
Ile Ser Ala Asp Phe Gln Gly Gly Phe Glu Pro Tyr Glu 20 25 30 Glu
Glu Asp Ala Glu Arg Ile Phe Arg Arg Ser Pro Thr Cys Ile Pro 35 40
45 Thr Gly Gln Pro Cys Pro Tyr Asn Glu Asn Cys Cys Asn Gln Ser Cys
50 55 60 Thr Tyr Lys Ala Asn Glu Asn Gly Asn Gln Val Lys Arg Cys
Asp 65 70 75 7437PRTHadronyche versuta 74Ser Pro Thr Cys Ile Pro
Thr Gly Gln Pro Cys Pro Tyr Asn Glu Asn 1 5 10 15 Cys Cys Asn Gln
Ser Cys Thr Tyr Lys Ala Asn Glu Asn Gly Asn Gln 20 25 30 Val Lys
Arg Cys Asp 35 7579PRTHadronyche versuta 75Met Asn Thr Ala Thr Gly
Phe Ile Val Leu Leu Val Leu Ala Thr Val 1 5 10 15 Ile Gly Cys Ile
Ser Ala Asp Phe Gln Gly Gly Phe Glu Pro Tyr Glu 20 25 30 Glu Glu
Asp Ala Glu Arg Ile Phe Arg Arg Ser Pro Thr Cys Ile Pro 35 40 45
Thr Gly Gln Pro Cys Pro Tyr Asn Glu Asn Cys Cys Ser Gln Ser Cys 50
55 60 Thr Tyr Lys Ala Asn Glu Asn Gly Asn Gln Val Lys Arg Cys Asp
65 70 75 7637PRTHadronyche versuta 76Ser Pro Thr Cys Ile Pro Thr
Gly Gln Pro Cys Pro Tyr Asn Glu Asn 1 5 10 15 Cys Cys Ser Gln Ser
Cys Thr Tyr Lys Ala Asn Glu Asn Gly Asn Gln 20 25 30 Val Lys Arg
Cys Asp 35 7779PRTHadronyche versuta 77Met Asn Thr Ala Thr Gly Phe
Ile Val Leu Leu Val Leu Ala Thr Val 1 5 10 15 Ile Gly Cys Ile Ser
Val Asp Phe Gln Gly Gly Phe Glu Ser Tyr Glu 20 25 30 Glu Glu Asp
Ala Glu Arg Ile Phe Arg Arg Ser Pro Thr Cys Ile Pro 35 40 45 Thr
Gly Gln Pro Cys Pro Tyr Asn Glu Asn Cys Cys Ser Gln Ser Cys 50 55
60 Thr Tyr Lys Ala Asn Glu Asn Gly Asn Gln Val Lys Arg Cys Asp 65
70 75 7837PRTHadronyche versuta 78Ser Pro Thr Cys Ile Pro Thr Gly
Gln Pro Cys Pro Tyr Asn Glu Asn 1 5 10 15 Cys Cys Ser Gln Ser Cys
Thr Tyr Lys Ala Asn Glu Asn Gly Asn Gln 20 25 30 Val Lys Arg Cys
Asp 35 7978PRTHadronyche versuta 79Met Asn Thr Ala Thr Gly Phe Ile
Val Leu Leu Val Leu Ala Thr Val 1 5 10 15 Ile Gly Cys Ile Ser Ala
Asp Phe Gln Gly Gly Phe Glu Ser Ser Val 20 25 30 Glu Asp Ala Glu
Arg Leu Phe Arg Arg Ser Ser Thr Cys Ile Arg Thr 35 40 45 Asp Gln
Pro Cys Pro Tyr Asn Glu Ser Cys Cys Ser Gly Ser Cys Thr 50 55 60
Tyr Lys Ala Asn Glu Asn Gly Asn Gln Val Lys Arg Cys Asp 65 70 75
8037PRTHadronyche versuta 80Ser Ser Thr Cys Ile Arg Thr Asp Gln Pro
Cys Pro Tyr Asn Glu Ser 1 5 10 15 Cys Cys Ser Gly Ser Cys Thr Tyr
Lys Ala Asn Glu Asn Gly Asn Gln 20 25 30 Val Lys Arg Cys Asp 35
8178PRTHadronyche versuta 81Met Asn Thr Ala Thr Gly Phe Ile Val Leu
Leu Val Leu Ala Thr Val 1 5 10 15 Ile Gly Cys Ile Ser Ala Asp Phe
Gln Gly Gly Phe Glu Pro Tyr Glu 20 25 30 Glu Glu Asp Ala Glu Arg
Ile Phe Arg Arg Ser Thr Cys Thr Pro Thr 35 40 45 Asp Gln Pro Cys
Pro Tyr His Glu Ser Cys Cys Ser Gly Ser Cys Thr 50 55 60 Tyr Lys
Ala Asn Glu Asn Gly Asn Gln Val Lys Arg Cys Asp 65 70 75
8236PRTHadronyche versuta 82Ser Thr Cys Thr Pro Thr Asp Gln Pro Cys
Pro Tyr His Glu Ser Cys 1 5 10 15 Cys Ser Gly Ser Cys Thr Tyr Lys
Ala Asn Glu Asn Gly Asn Gln Val 20 25 30 Lys Arg Cys Asp 35
8378PRTHadronyche versuta 83Met Asn Thr Ala Thr Gly Phe Ile Val Leu
Leu Val Leu Ala Thr Val 1 5 10 15 Ile Gly Cys Ile Ser Ala Asp Phe
Glu Gly Ser Phe Glu Pro Tyr Glu 20 25 30 Glu Glu Asp Ala Glu Arg
Ile Phe Arg Arg Ser Thr Cys Thr Pro Thr 35 40 45 Asp Gln Pro Cys
Pro Tyr Asp Glu Ser Cys Cys Ser Gly Ser Cys Thr 50 55 60 Tyr Lys
Ala Asn Glu Asn Gly Asn Gln Val Lys Arg Cys Asp 65 70 75
8436PRTHadronyche versuta 84Ser Thr Cys Thr Pro Thr Asp Gln Pro Cys
Pro Tyr Asp Glu Ser Cys 1 5 10 15 Cys Ser Gly Ser Cys Thr Tyr Lys
Ala Asn Glu Asn Gly Asn Gln Val 20 25 30 Lys Arg Cys Asp 35
8578PRTHadronyche versuta 85Met Asn Thr Ala Thr Gly Phe Ile Val Leu
Leu Val Leu Ala Thr Val 1 5 10 15 Ile Gly Cys Ile Ser Ala Asp Phe
Gln Gly Ser Phe Glu Pro Tyr Glu 20 25 30 Glu Glu Asp Ala Glu Arg
Ile Phe Arg Arg Ser Thr Cys Thr Pro Thr 35 40 45 Asp Gln Pro Cys
Pro Tyr Asp Glu Ser Cys Cys Ser Gly Ser Cys Thr 50 55 60 Tyr Lys
Ala Asn Glu Asn Gly Asn Gln Val Lys Arg Cys Asp 65 70 75
8636PRTHadronyche versuta 86Ser Thr Cys Thr Pro Thr Asp Gln Pro Cys
Pro Tyr Asp Glu Ser Cys 1 5 10 15 Cys Ser Gly Ser Cys Thr Tyr Lys
Ala Asn Glu Asn Gly Asn Gln Val 20 25 30 Lys Arg Cys Asp 35
8778PRTHadronyche versuta 87Met Asn Thr Ala Thr Gly Phe Ile Val Leu
Leu Val Leu Ala Thr Val 1 5 10 15 Ile Gly Cys Ile Ser Ala Asp Phe
Gln Gly Ser Phe Glu Pro Tyr Glu 20 25 30 Glu Glu Asp Ala Glu Arg
Ile Phe Arg Arg Ser Thr Cys Thr Pro Thr 35 40 45 Asp Gln Pro Cys
Pro Tyr His Glu Ser Cys Cys Ser Gly Ser Cys Thr 50 55 60 Tyr Lys
Ala Asn Glu Asn Gly Asn Gln Val Lys Arg Cys Asp 65 70 75
8836PRTHadronyche versuta 88Ser Thr Cys Thr Pro Thr Asp Gln Pro Cys
Pro Tyr His Glu Ser Cys 1 5 10 15 Cys Ser Gly Ser Cys Thr Tyr Lys
Ala Asn Glu Asn Gly Asn Gln Val 20 25 30 Lys Arg Cys Asp 35
8978PRTHadronyche versuta 89Met Asn Thr Ala Thr Gly Phe Ile Val Leu
Leu Val Leu Ala Thr Val 1 5 10 15 Ile Gly Cys Ile Ser Ala Asp Phe
Gln Gly Gly Phe Glu Pro Tyr Glu 20 25 30 Glu Glu Asp Ala Glu Arg
Ile Phe Arg Arg Ser Thr Cys Thr Pro Thr 35 40 45 Asp Gln Pro Cys
Pro Tyr Asp Glu Ser Cys Cys Ser Gly Ser Cys Thr 50 55 60 Tyr Lys
Ala Asn Glu Asn Gly Asn Gln Val Lys Arg Cys Asp 65 70 75
9036PRTHadronyche versuta 90Ser Thr Cys Thr Pro Thr Asp Gln Pro Cys
Pro Tyr Asp Glu Ser Cys 1 5 10 15 Cys Ser Gly Ser Cys Thr Tyr Lys
Ala Asn Glu Asn Gly Asn Gln Val 20 25 30 Lys Arg Cys Asp 35
9136PRTAtrax infensus 91Ser Thr Cys Thr Pro Thr Asp Gln Pro Cys Pro
Tyr His Glu Ser Cys 1 5 10 15 Cys Ser Gly Ser Cys Thr Tyr Lys Ala
Asn Glu Asn Gly Asn Gln Val 20 25 30 Lys Arg Cys Asp 35
9237PRTAtrax infensus 92Ser Pro Thr Cys Ile Pro Thr Gly Gln Pro Cys
Pro Tyr Asn Glu Asn 1 5 10 15 Cys Cys Ser Gln Ser Cys Thr Tyr Lys
Ala Asn Glu Asn Gly Asn Gln 20 25 30 Val Lys Arg Cys Asp 35
9337PRTAtrax infensus 93Ser Ser Thr Cys Ile Arg Thr Asp Gln Pro Cys
Pro Tyr Asn Glu Ser 1 5 10 15 Cys Cys Ser Gly Ser Cys Thr Tyr Lys
Ala Asn Glu Asn Gly Asn Gln 20 25 30 Val Lys Arg Cys Asp 35
9437PRTAtrax robustus 94Ser Ser Val Cys Ile Pro Ser Gly Gln Pro Cys
Pro Tyr Asn Glu His 1 5 10 15 Cys Cys Ser Gly Ser Cys Thr Tyr Lys
Glu Asn Glu Asn Gly Asn Thr 20 25 30 Val Gln Arg Cys Asp 35
9537PRTHadronyche versuta 95Ser Pro Thr Cys Ile Pro Ser Gly Gln Pro
Cys Pro Tyr Asn Glu Asn 1 5
10 15 Cys Cys Ser Gln Ser Cys Thr Phe Lys Glu Asn Glu Asn Gly Asn
Thr 20 25 30 Val Lys Arg Cys Asp 35 9637PRTAtrax formidabillis
96Ser Pro Thr Cys Thr Gly Ala Asp Arg Pro Cys Ala Ala Cys Cys Pro 1
5 10 15 Cys Cys Pro Gly Thr Ser Cys Lys Gly Pro Glu Pro Asn Gly Val
Ser 20 25 30 Tyr Cys Arg Asn Asp 35 9736PRTAtrax formidabillis
97Ser Pro Thr Cys Thr Gly Ala Asp Arg Pro Cys Ala Ala Cys Cys Pro 1
5 10 15 Cys Cys Pro Gly Thr Ser Cys Lys Gly Pro Glu Pro Asn Gly Val
Ser 20 25 30 Tyr Cys Arg Asn 35 9837PRTAtrax formidabillis 98Ser
Pro Thr Cys Ile Arg Ser Gly Gln Pro Cys Pro Tyr Asn Glu Asn 1 5 10
15 Cys Cys Ser Gln Ser Cys Thr Phe Lys Thr Asn Glu Asn Gly Asn Thr
20 25 30 Val Lys Arg Cys Asp 35 999PRTAtrax infensus 99Asn Gly Asn
Gln Val Lys Arg Cys Asp 1 5 1009PRTAtrax infensus 100Asn Gly Asn
Gln Val Lys Arg Cys Asp 1 5 1019PRTHadronyche versutus 101Asn Gly
Asn Thr Val Lys Arg Cys Asp 1 5 10215PRTHadronyche versutus 102Ser
Pro Thr Cys Ile Pro Ser Gly Gln Pro Cys Pro Tyr Asn Glu 1 5 10 15
10316PRTAgelenopsis aperta 103Glu Cys Val Pro Glu Asn Gly His Cys
Arg Asp Trp Tyr Asp Glu Cys 1 5 10 15 10437PRTAgelenopsis aperta
104Glu Cys Ala Thr Lys Asn Lys Arg Cys Ala Asp Trp Ala Gly Pro Trp
1 5 10 15 Cys Cys Asp Gly Leu Tyr Cys Ser Cys Arg Ser Tyr Pro Gly
Cys Met 20 25 30 Cys Arg Pro Ser Ser 35 10538PRTAgelenopsis aperta
105Ala Asp Cys Val Gly Asp Gly Gln Arg Cys Ala Asp Trp Ala Gly Pro
1 5 10 15 Tyr Cys Cys Ser Gly Tyr Tyr Cys Ser Cys Arg Ser Met Pro
Tyr Cys 20 25 30 Arg Cys Arg Ser Asp Ser 35 10637PRTAgelenopsis
aperta 106Ala Cys Val Gly Glu Asn Gln Gln Cys Ala Asp Trp Ala Gly
Pro His 1 5 10 15 Cys Cys Asp Gly Tyr Tyr Cys Thr Cys Arg Tyr Phe
Pro Lys Cys Ile 20 25 30 Cys Arg Asn Asn Asn 35 10737PRTAgelenopsis
aperta 107Ala Cys Val Gly Glu Asn Lys Gln Cys Ala Asp Trp Ala Gly
Pro His 1 5 10 15 Cys Cys Asp Gly Tyr Tyr Cys Thr Cys Arg Tyr Phe
Pro Lys Cys Ile 20 25 30 Cys Arg Asn Asn Asn 35 10837PRTAgelenopsis
aperta 108Asp Cys Val Gly Glu Ser Gln Gln Cys Ala Asp Trp Ala Gly
Pro His 1 5 10 15 Cys Cys Asp Gly Tyr Tyr Cys Thr Cys Arg Tyr Phe
Pro Lys Cys Ile 20 25 30 Cys Val Asn Asn Asn 35 10936PRTHololena
curta 109Ser Cys Val Gly Glu Tyr Gly Arg Cys Arg Ser Ala Tyr Glu
Asp Cys 1 5 10 15 Cys Asp Gly Tyr Tyr Cys Asn Cys Ser Gln Pro Pro
Tyr Cys Leu Cys 20 25 30 Arg Asn Asn Asn 35 11038PRTHololena curta
110Ala Asp Cys Val Gly Asp Gly Gln Lys Cys Ala Asp Trp Phe Gly Pro
1 5 10 15 Tyr Cys Cys Ser Gly Tyr Tyr Cys Ser Cys Arg Ser Met Pro
Tyr Cys 20 25 30 Arg Cys Arg Ser Asp Ser 35 11170PRTAndroctonus
australis Hector 111Lys Lys Asn Gly Tyr Ala Val Asp Ser Ser Gly Lys
Ala Pro Glu Cys 1 5 10 15 Leu Leu Ser Asn Tyr Cys Asn Asn Gln Cys
Thr Lys Val His Tyr Ala 20 25 30 Asp Lys Gly Tyr Cys Cys Leu Leu
Ser Cys Tyr Cys Phe Gly Leu Asn 35 40 45 Asp Asp Lys Lys Val Leu
Glu Ile Ser Asp Thr Arg Lys Ser Tyr Cys 50 55 60 Asp Thr Thr Ile
Ile Asn 65 70 11270PRTAndroctonus australis Hector 112Lys Lys Asn
Gly Tyr Ala Val Asp Ser Ser Gly Lys Ala Pro Glu Cys 1 5 10 15 Leu
Leu Ser Asn Tyr Cys Asn Asn Glu Cys Thr Lys Val His Tyr Ala 20 25
30 Asp Lys Gly Tyr Cys Cys Leu Leu Ser Cys Tyr Cys Phe Gly Leu Asn
35 40 45 Asp Asp Lys Lys Val Leu Glu Ile Ser Asp Thr Arg Lys Ser
Tyr Cys 50 55 60 Asp Thr Thr Ile Ile Asn 65 70 11370PRTAndroctonus
australis Hector 113Lys Lys Asp Gly Tyr Ala Val Asp Ser Ser Gly Lys
Ala Pro Glu Cys 1 5 10 15 Leu Leu Ser Asn Tyr Cys Tyr Asn Glu Cys
Thr Lys Val His Tyr Ala 20 25 30 Asp Lys Gly Tyr Cys Cys Leu Leu
Ser Cys Tyr Cys Phe Gly Leu Asn 35 40 45 Asp Asp Lys Lys Val Leu
Glu Ile Ser Asp Thr Arg Lys Ser Tyr Cys 50 55 60 Asp Thr Pro Ile
Ile Asn 65 70 11433PRTScorpio maurus palmatus 114Ala Leu Pro Leu
Ser Gly Glu Tyr Glu Pro Cys Val Arg Pro Arg Lys 1 5 10 15 Cys Lys
Pro Gly Leu Val Cys Asn Lys Gln Gln Ile Cys Val Asp Pro 20 25 30
Lys 11561PRTLeiurus quinquestriatus 115Asp Gly Tyr Ile Arg Lys Arg
Asp Gly Cys Lys Leu Ser Cys Leu Phe 1 5 10 15 Gly Asn Glu Gly Cys
Asn Lys Glu Cys Lys Ser Tyr Gly Gly Ser Tyr 20 25 30 Gly Tyr Cys
Trp Thr Trp Gly Leu Ala Cys Trp Cys Glu Gly Leu Pro 35 40 45 Asp
Glu Lys Thr Trp Lys Ser Glu Thr Asn Thr Cys Gly 50 55 60
11661PRTButhotus judaicus 116Asp Gly Tyr Ile Arg Lys Lys Asp Gly
Cys Lys Val Ser Cys Ile Ile 1 5 10 15 Gly Asn Glu Gly Cys Arg Lys
Glu Cys Val Ala His Gly Gly Ser Phe 20 25 30 Gly Tyr Cys Trp Thr
Trp Gly Leu Ala Cys Trp Cys Glu Asn Leu Pro 35 40 45 Asp Ala Val
Thr Trp Lys Ser Ser Thr Asn Thr Cys Gly 50 55 60 11739PRTAtrax
robustus 117Gly Ser Ser Pro Thr Cys Ile Pro Ser Gly Gln Pro Cys Pro
Tyr Asn 1 5 10 15 Glu Asn Cys Cys Ser Gln Ser Cys Thr Phe Lys Glu
Asn Glu Asn Gly 20 25 30 Asn Thr Val Lys Arg Cys Asp 35
11839PRTHadronyche Versuta 118Gly Ser Ala Ile Cys Thr Gly Ala Asp
Arg Pro Cys Ala Ala Cys Cys 1 5 10 15 Pro Cys Cys Pro Gly Thr Ser
Cys Lys Ala Glu Ser Asn Gly Val Ser 20 25 30 Tyr Cys Arg Lys Asp
Glu Pro 35 11941PRTHadronyche Versuta 119Gly Ser Gln Tyr Cys Val
Pro Val Asp Gln Pro Cys Ser Leu Asn Thr 1 5 10 15 Gln Pro Cys Cys
Asp Asp Ala Thr Cys Thr Gln Glu Arg Asn Glu Asn 20 25 30 Gly His
Thr Val Tyr Tyr Cys Arg Ala 35 40 12060PRTHadronyche versuta 120Met
Asn Thr Ala Thr Gly Phe Ile Val Leu Leu Val Leu Ala Thr Val 1 5 10
15 Leu Gly Gly Val Glu Ala Gly Glu Ser His Met Arg Lys Asp Ala Met
20 25 30 Gly Arg Val Arg Arg Gln Tyr Cys Val Pro Val Asp Gln Pro
Cys Ser 35 40 45 Leu Asn Thr Gln Pro Cys Cys Asp Asp Ala Thr Cys 50
55 60 12139PRTHadronyche versuta 121Gln Tyr Cys Val Pro Val Asp Gln
Pro Cys Ser Leu Asn Thr Gln Pro 1 5 10 15 Cys Cys Asp Asp Ala Tyr
Cys Thr Gln Glu Arg Asn Glu Asn Gly His 20 25 30 Thr Val Tyr Tyr
Cys Arg Ala 35 12239PRTHadronyche versuta 122Gly Ser Cys Val Pro
Val Asp Gln Pro Cys Ser Leu Asn Thr Gln Pro 1 5 10 15 Cys Cys Asp
Asp Ala Thr Cys Thr Gln Glu Arg Asn Glu Asn Gly His 20 25 30 Thr
Val Tyr Tyr Cys Arg Ala 35 12374PRTHadronyche versuta 123Met Asn
Thr Ala Thr Gly Phe Ile Val Leu Leu Val Leu Ala Thr Ile 1 5 10 15
Leu Gly Gly Ile Glu Ala Gly Glu Ser His Met Arg Lys Asp Ala Met 20
25 30 Gly Arg Val Arg Arg Gln Tyr Cys Val Pro Val Asp Gln Pro Cys
Ser 35 40 45 Leu Asn Thr Gln Pro Cys Cys Asp Asp Ala Thr Cys Thr
Glu Arg Asn 50 55 60 Glu Asn Gly His Thr Val Tyr Tyr Cys Arg 65 70
12438PRTHadronyche versuta 124Gln Tyr Cys Val Pro Val Asp Gln Pro
Cys Ser Leu Asn Thr Gln Pro 1 5 10 15 Cys Cys Asp Asp Ala Thr Cys
Thr Gln Glu Arg Asn Glu Asn Gly His 20 25 30 Thr Val Tyr Tyr Cys
Arg 35 12575PRTHadronyche versuta 125Met Asn Thr Ala Thr Gly Phe
Ile Val Leu Leu Val Leu Ala Thr Val 1 5 10 15 Leu Gly Gly Ile Glu
Ala Gly Glu Ser His Met Arg Lys Asp Ala Met 20 25 30 Gly Arg Val
Arg Arg Gln Tyr Cys Val Pro Val Asp Gln Pro Cys Ser 35 40 45 Leu
Asn Thr Gln Pro Cys Cys Asp Asp Ala Tyr Cys Thr Gln Glu Leu 50 55
60 Asn Glu Asn Asp Asn Thr Val Tyr Tyr Cys Arg 65 70 75
12638PRTHadronyche versuta 126Gln Tyr Cys Val Pro Val Asp Gln Pro
Cys Ser Leu Asn Thr Gln Pro 1 5 10 15 Cys Cys Asp Asp Ala Thr Cys
Thr Gln Glu Leu Asn Glu Asn Asp Asn 20 25 30 Thr Val Tyr Tyr Cys
Arg 35 12776PRTHadronyche versuta 127Met Asn Thr Ala Thr Gly Phe
Ile Val Leu Leu Val Leu Ala Thr Ile 1 5 10 15 Leu Gly Gly Ile Glu
Ala Gly Glu Ser His Met Arg Lys Asp Ala Met 20 25 30 Gly Arg Val
Arg Arg Gln Tyr Cys Val Pro Val Asp Gln Pro Cys Ser 35 40 45 Leu
Asn Thr Gln Pro Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Arg 50 55
60 Asn Glu Asn Gly His Thr Val Tyr Tyr Cys Arg Ala 65 70 75
12839PRTHadronyche versuta 128Gln Tyr Cys Val Pro Val Asp Gln Pro
Cys Ser Leu Asn Thr Gln Pro 1 5 10 15 Cys Cys Asp Asp Ala Thr Cys
Thr Gln Glu Arg Asn Glu Asn Gly His 20 25 30 Thr Val Tyr Tyr Cys
Arg Ala 35 12976PRTHadronyche versuta 129Met Asn Thr Ala Thr Gly
Phe Ile Val Leu Leu Val Leu Ala Thr Val 1 5 10 15 Leu Gly Gly Ile
Glu Ala Gly Glu Ser His Met Arg Lys Asp Ala Met 20 25 30 Gly Arg
Val Arg Arg Gln Tyr Cys Val Pro Val Asp Gln Pro Cys Ser 35 40 45
Leu Asn Thr Gln Pro Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Leu 50
55 60 Asn Glu Asn Ala Asn Pro Val Tyr Tyr Cys Arg Ala 65 70 75
13039PRTHadronyche versuta 130Gln Tyr Cys Val Pro Val Asp Gln Pro
Cys Ser Leu Asn Thr Gln Pro 1 5 10 15 Cys Cys Asp Asp Ala Thr Cys
Thr Gln Glu Leu Asn Glu Asn Ala Asn 20 25 30 Pro Val Tyr Tyr Cys
Arg Ala 35 13176PRTHadronyche versuta 131Met Asn Thr Thr Thr Gly
Phe Ile Val Leu Leu Val Leu Ala Thr Ile 1 5 10 15 Leu Gly Gly Ile
Glu Ala Gly Glu Ser His Met Arg Lys Asp Ala Met 20 25 30 Gly Arg
Val Arg Arg Gln Tyr Cys Val Pro Val Asp Gln Pro Cys Ser 35 40 45
Leu Asn Thr Gln Pro Cys Cys Asp Asp Ala Tyr Cys Thr Gln Glu Leu 50
55 60 Asn Glu Asn Asp Asn Thr Val Tyr Tyr Cys Arg Ala 65 70 75
13239PRTHadronyche versuta 132Gln Tyr Cys Val Pro Val Asp Gln Pro
Cys Ser Leu Asn Thr Gln Pro 1 5 10 15 Cys Cys Asp Asp Ala Thr Cys
Thr Gln Glu Leu Asn Glu Asn Asp Asn 20 25 30 Thr Val Tyr Tyr Cys
Arg Ala 35 13376PRTHadronyche versuta 133Met Asn Thr Ala Thr Gly
Phe Ile Val Leu Leu Val Leu Ala Thr Val 1 5 10 15 Leu Gly Gly Ile
Glu Ala Gly Glu Ser His Met Arg Lys Asp Ala Met 20 25 30 Gly Arg
Val Arg Arg Gln Tyr Cys Val Pro Val Asp Gln Pro Cys Ser 35 40 45
Leu Asn Thr Gln Pro Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Leu 50
55 60 Asn Glu Asn Asp Asn Thr Val Tyr Tyr Cys Arg Ala 65 70 75
13439PRTHadronyche versuta 134Gln Tyr Cys Val Pro Val Asp Gln Pro
Cys Ser Leu Asn Thr Gln Pro 1 5 10 15 Cys Cys Asp Asp Ala Thr Cys
Thr Gln Glu Leu Asn Glu Asn Asp Asn 20 25 30 Thr Val Tyr Tyr Cys
Arg Ala 35 13576PRTHadronyche versuta 135Met Asn Thr Ala Thr Gly
Phe Ile Val Phe Leu Val Leu Ala Thr Val 1 5 10 15 Leu Gly Gly Ile
Glu Ala Gly Glu Ser His Met Arg Lys Asp Ala Met 20 25 30 Gly Arg
Val Arg Arg Gln Tyr Cys Val Pro Val Asp Gln Pro Cys Ser 35 40 45
Leu Asn Thr Gln Pro Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Leu 50
55 60 Asn Glu Asn Asp Asn Thr Val Tyr Tyr Cys Arg Ala 65 70 75
13639PRTHadronyche versuta 136Gln Tyr Cys Val Pro Val Asp Gln Pro
Cys Ser Leu Asn Thr Gln Pro 1 5 10 15 Cys Cys Asp Asp Ala Thr Cys
Thr Gln Glu Leu Asn Glu Asn Asp Asn 20 25 30 Thr Val Tyr Tyr Cys
Arg Ala 35 13776PRTAtrax robustus 137Met Asn Thr Ala Thr Gly Phe
Ile Val Leu Leu Val Leu Ala Thr Val 1 5 10 15 Leu Gly Gly Ile Glu
Ala Arg Glu Ser His Met Arg Lys Asp Ala Met 20 25 30 Gly Arg Val
Arg Arg Gln Tyr Cys Val Pro Val Asp Gln Pro Cys Ser 35 40 45 Leu
Asn Thr Gln Pro Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Leu 50 55
60 Asn Glu Asn Asp Asn Thr Val Tyr Tyr Cys Arg Ala 65 70 75
13839PRTAtrax robustus 138Gln Tyr Cys Val Pro Val Asp Gln Pro Cys
Ser Leu Asn Thr Gln Pro 1 5 10 15 Cys Cys Asp Asp Ala Thr Cys Thr
Gln Glu Leu Asn Glu Asn Asp Asn 20 25 30 Thr Val Tyr Tyr Cys Arg
Ala 35 13922PRTHadronyche versutaMISC_FEATURE(4)..(4)Xaa can be any
naturally occuring amino acid 139Met Asn Thr Xaa Thr Gly Phe Ile
Val Xaa Leu Val Leu Ala Thr Xaa 1 5 10 15 Leu Gly Gly Xaa Glu Ala
20 14015PRTHadronyche versutaMISC_FEATURE(1)..(1)Xaa can be any
naturally occuring amino acid 140Xaa Glu Ser His Met Arg Lys Asp
Ala Met Gly Arg Val Arg Arg 1 5 10 15 14164PRTLeiurus
quinquestriatus hebraeus 141Val Arg Asp Ala Tyr Ile Ala Lys Asn Tyr
Asn Cys Val Tyr Glu Cys 1 5 10 15 Phe Arg Asp Ala Tyr Cys Asn Glu
Leu Cys Thr Lys Asn Gly Ala Ser 20 25 30 Ser Gly Tyr Cys Gln Trp
Ala Gly Lys Tyr Gly Asn Ala Cys Trp Cys 35 40 45 Tyr Ala Leu Pro
Asp Asn Val Pro Ile Arg Val Pro Gly Lys Cys Arg 50 55
60 14264PRTLeiurus quinquestriatus quinquestriatus 142Val Arg Asp
Ala Tyr Ile Ala Lys Asn Tyr Asn Cys Val Tyr Glu Cys 1 5 10 15 Phe
Arg Asp Ser Tyr Cys Asn Asp Leu Cys Thr Lys Asn Gly Ala Ser 20 25
30 Ser Gly Tyr Cys Gln Trp Ala Gly Lys Tyr Gly Asn Ala Cys Trp Cys
35 40 45 Tyr Ala Leu Pro Asp Asn Val Pro Ile Arg Val Pro Gly Lys
Cys His 50 55 60 14365PRTBothus occitanus tunetanus 143Val Arg Asp
Ala Tyr Ile Ala Gln Asn Tyr Asn Cys Val Tyr Phe Cys 1 5 10 15 Met
Lys Asp Asp Tyr Cys Asn Asp Leu Cys Thr Lys Asn Gly Ala Ser 20 25
30 Ser Gly Tyr Cys Gln Trp Ala Gly Lys Tyr Gly Asn Ala Cys Trp Cys
35 40 45 Tyr Ala Leu Pro Asp Asn Val Pro Ile Arg Ile Pro Gly Lys
Cys His 50 55 60 Ser 65 14464PRTHottentotta judaica 144Gly Arg Asp
Ala Tyr Ala Leu Asp Asn Leu Asn Cys Ala Tyr Thr Cys 1 5 10 15 Gly
Ser Lys Ser Tyr Cys Asn Thr Glu Cys Thr Lys Asn Gly Ala Val 20 25
30 Ser Gly Tyr Cys Gln Trp Leu Gly Lys Tyr Gly Asn Ala Cys Trp Cys
35 40 45 Ile Asn Leu Pro Asp Lys Val Pro Ile Arg Ile Pro Gly Ala
Cys Arg 50 55 60 14567PRTLeiurus quinquestriatus hebraeus 145Val
Arg Asp Gly Tyr Ile Ala Gln Pro Glu Asn Cys Val Tyr His Cys 1 5 10
15 Phe Pro Gly Ser Ser Gly Cys Asp Thr Leu Cys Lys Glu Lys Gly Gly
20 25 30 Thr Ser Gly His Cys Gly Phe Lys Val Gly His Gly Leu Ala
Cys Trp 35 40 45 Cys Asn Ala Leu Pro Asp Asn Val Gly Ile Ile Val
Glu Gly Glu Lys 50 55 60 Cys His Ser 65 14666PRTButhus occitanus
mardochei 146Gly Arg Asp Gly Tyr Ile Ala Gln Pro Glu Asn Cys Val
Tyr His Cys 1 5 10 15 Phe Pro Gly Ser Ser Gly Cys Asp Thr Leu Cys
Lys Glu Lys Gly Ala 20 25 30 Thr Ser Gly His Cys Gly Phe Leu Pro
Gly Ser Gly Val Ala Cys Trp 35 40 45 Cys Asp Asn Leu Pro Asn Lys
Val Pro Ile Val Val Gly Gly Glu Lys 50 55 60 Cys His 65
14765PRTButhus occitanus mardochei 147Gly Arg Asp Ala Tyr Ile Ala
Gln Pro Glu Asn Cys Val Tyr Glu Cys 1 5 10 15 Ala Lys Asn Ser Tyr
Cys Asn Asp Leu Cys Thr Lys Asn Gly Ala Lys 20 25 30 Ser Gly Tyr
Cys Gln Trp Leu Gly Lys Tyr Gly Asn Ala Cys Trp Cys 35 40 45 Glu
Asp Leu Pro Asp Asn Val Pro Ile Arg Ile Pro Gly Lys Cys His 50 55
60 Phe 65 14864PRTBothus martensii Karsch 148Val Arg Asp Ala Tyr
Ile Ala Lys Pro His Asn Cys Val Tyr Glu Cys 1 5 10 15 Ala Arg Asn
Glu Tyr Cys Asn Asp Leu Cys Thr Lys Asn Gly Ala Lys 20 25 30 Ser
Gly Tyr Cys Gln Trp Val Gly Lys Tyr Gly Asn Gly Cys Trp Cys 35 40
45 Ile Glu Leu Pro Asp Asn Val Pro Ile Arg Val Pro Gly Lys Cys His
50 55 60 14964PRTBothus martensii Karsch 149Val Arg Asp Ala Tyr Ile
Ala Lys Pro His Asn Cys Val Tyr Ser Cys 1 5 10 15 Ala Arg Asn Glu
Trp Cys Asn Asp Leu Cys Thr Lys Asn Gly Ala Lys 20 25 30 Ser Gly
Tyr Cys Gln Trp Val Gly Lys Tyr Gly Asn Gly Cys Trp Cys 35 40 45
Ile Glu Leu Pro Asp Asn Val Pro Ile Arg Val Pro Gly Lys Cys His 50
55 60 15064PRTBothus martensii Karsch 150Val Arg Asp Ala Tyr Ile
Ala Lys Pro Glu Asn Cys Val Tyr His Cys 1 5 10 15 Ala Gly Asn Glu
Gly Cys Asn Lys Leu Cys Thr Asp Asn Gly Ala Glu 20 25 30 Ser Gly
Tyr Cys Gln Trp Gly Gly Arg Tyr Gly Asn Ala Cys Trp Cys 35 40 45
Ile Lys Leu Pro Asp Asp Val Pro Ile Arg Val Pro Gly Lys Cys His 50
55 60 15166PRTBothus martensii Karsch 151Val Arg Asp Gly Tyr Ile
Ala Leu Pro His Asn Cys Ala Tyr Gly Cys 1 5 10 15 Leu Asn Asn Glu
Tyr Cys Asn Asn Leu Cys Thr Lys Asp Gly Ala Lys 20 25 30 Ile Gly
Tyr Cys Asn Ile Val Gly Lys Tyr Gly Asn Ala Cys Trp Cys 35 40 45
Ile Gln Leu Pro Asp Asn Val Pro Ile Arg Val Pro Gly Arg Cys His 50
55 60 Pro Ala 65 15264PRTLeiurus quinquestriatus 152Val Arg Asp Gly
Tyr Ile Ala Gln Pro Glu Asn Cys Val Tyr His Cys 1 5 10 15 Ile Pro
Asp Cys Asp Thr Leu Cys Lys Asp Asn Gly Gly Thr Gly Gly 20 25 30
His Cys Gly Phe Lys Leu Gly His Gly Ile Ala Cys Trp Cys Asn Ala 35
40 45 Leu Pro Asp Asn Val Gly Ile Ile Val Asp Gly Val Lys Cys His
Lys 50 55 60 15366PRTLeiurus quinquestriatus 153Val Arg Asp Gly Tyr
Ile Ala Lys Pro Glu Asn Cys Ala His His Cys 1 5 10 15 Phe Pro Gly
Ser Ser Gly Cys Asp Thr Leu Cys Lys Glu Asn Gly Gly 20 25 30 Thr
Gly Gly His Cys Gly Phe Lys Val Gly His Gly Thr Ala Cys Trp 35 40
45 Cys Asn Ala Leu Pro Asp Lys Val Gly Ile Ile Val Asp Gly Val Lys
50 55 60 Cys His 65 15466PRTCentruroides noxius 154Lys Glu Gly Tyr
Leu Val Asp Ile Lys Asn Thr Gly Cys Lys Tyr Glu 1 5 10 15 Cys Leu
Lys Leu Gly Asp Asn Asp Tyr Cys Leu Arg Glu Cys Lys Gln 20 25 30
Gln Tyr Gly Lys Gly Ala Gly Gly Tyr Cys Tyr Ala Phe Ala Cys Trp 35
40 45 Cys Thr His Leu Tyr Glu Gln Ala Ile Val Trp Pro Leu Pro Asn
Lys 50 55 60 Arg Cys 65 15566PRTCentruroides noxius 155Lys Glu Gly
Tyr Leu Val Glu Leu Gly Thr Gly Cys Lys Tyr Glu Cys 1 5 10 15 Phe
Lys Leu Gly Asp Asn Asp Tyr Cys Leu Arg Glu Cys Lys Ala Arg 20 25
30 Tyr Gly Lys Gly Ala Gly Gly Tyr Cys Tyr Ala Phe Gly Cys Trp Cys
35 40 45 Thr Gln Leu Tyr Glu Gln Ala Val Val Trp Pro Leu Lys Asn
Lys Thr 50 55 60 Cys Arg 65 15666PRTCentruroides suffusus suffusus
156Lys Glu Gly Tyr Leu Val Ser Lys Ser Thr Gly Cys Lys Tyr Glu Cys
1 5 10 15 Leu Lys Leu Gly Asp Asn Asp Tyr Cys Leu Arg Glu Cys Lys
Gln Gln 20 25 30 Tyr Gly Lys Ser Ser Gly Gly Tyr Cys Tyr Ala Phe
Ala Cys Trp Cys 35 40 45 Thr His Leu Tyr Glu Gln Ala Val Val Trp
Pro Leu Pro Asn Lys Thr 50 55 60 Cys Asn 65 15766PRTCentruroides
suffusus suffusus 157Lys Glu Gly Tyr Leu Val Asn Ser Tyr Thr Gly
Cys Lys Phe Glu Cys 1 5 10 15 Phe Lys Leu Gly Asp Asn Asp Tyr Cys
Leu Arg Glu Cys Arg Gln Gln 20 25 30 Tyr Gly Lys Gly Ser Gly Gly
Tyr Cys Tyr Ala Phe Gly Cys Trp Cys 35 40 45 Thr His Leu Tyr Glu
Gln Ala Val Val Trp Pro Leu Pro Asn Lys Thr 50 55 60 Cys Asn 65
15876PRTButhotus judaicus 158Lys Lys Asn Gly Tyr Pro Leu Asp Arg
Asn Gly Lys Thr Thr Glu Cys 1 5 10 15 Ser Gly Val Asn Ala Ile Ala
Pro His Tyr Cys Asn Ser Glu Cys Thr 20 25 30 Lys Val Tyr Val Ala
Glu Ser Gly Tyr Cys Cys Trp Gly Ala Cys Tyr 35 40 45 Cys Phe Gly
Leu Glu Asp Asp Lys Pro Ile Gly Pro Met Lys Asp Ile 50 55 60 Thr
Lys Lys Tyr Cys Asp Val Gln Ile Ile Pro Ser 65 70 75
15970PRTAndroctonus australis Hector 159Lys Lys Asn Gly Tyr Ala Val
Asp Ser Ser Gly Lys Ala Pro Glu Cys 1 5 10 15 Leu Leu Ser Asn Tyr
Cys Asn Asn Glu Cys Thr Lys Val His Tyr Ala 20 25 30 Asp Lys Gly
Tyr Cys Cys Leu Leu Ser Cys Tyr Cys Phe Gly Leu Asn 35 40 45 Asp
Asp Lys Lys Val Leu Glu Ile Ser Asp Thr Arg Lys Ser Tyr Cys 50 55
60 Asp Thr Thr Ile Ile Asn 65 70 16070PRTLeiurus quinquestriatus
quinquestriatus 160Lys Lys Asn Gly Tyr Ala Val Asp Ser Ser Gly Lys
Ala Pro Glu Cys 1 5 10 15 Leu Leu Ser Asn Tyr Cys Tyr Asn Glu Cys
Thr Lys Val His Tyr Ala 20 25 30 Asp Lys Gly Tyr Cys Cys Leu Leu
Ser Cys Tyr Cys Val Gly Leu Ser 35 40 45 Asp Asp Lys Lys Val Leu
Glu Ile Ser Asp Ala Arg Lys Lys Tyr Cys 50 55 60 Asp Phe Val Thr
Ile Asn 65 70 16170PRTLeiurus quinquestriatus hebraeus 161Lys Lys
Asn Gly Phe Ala Val Asp Ser Asn Gly Lys Ala Pro Glu Cys 1 5 10 15
Phe Phe Asp His Tyr Cys Asn Ser Glu Cys Thr Lys Val Tyr Tyr Ala 20
25 30 Glu Lys Gly Tyr Cys Cys Leu Leu Ser Cys Tyr Cys Phe Gly Leu
Asn 35 40 45 Asp Asp Lys Lys Val Leu Glu Ile Ser Asp Thr Thr Lys
Lys Tyr Cys 50 55 60 Asp Phe Thr Ile Ile Asn 65 70 16272PRTBothus
martensii Karsch 162Lys Lys Asn Gly Tyr Ala Val Asp Ser Ser Gly Lys
Val Ala Glu Cys 1 5 10 15 Leu Phe Asn Asn Tyr Cys Asn Asn Glu Cys
Thr Lys Val Tyr Tyr Ala 20 25 30 Asp Lys Gly Tyr Cys Cys Leu Leu
Lys Cys Tyr Cys Phe Gly Leu Leu 35 40 45 Asp Asp Lys Pro Val Leu
Asp Ile Trp Asp Ser Thr Lys Asn Tyr Cys 50 55 60 Asp Val Gln Ile
Ile Asp Leu Ser 65 70 16361PRTLeiurus quinquestriatus hebraeus
163Asp Gly Tyr Ile Lys Arg Arg Asp Gly Cys Lys Val Ala Cys Leu Ile
1 5 10 15 Gly Asn Glu Gly Cys Asp Lys Glu Cys Lys Ala Tyr Gly Gly
Ser Tyr 20 25 30 Gly Tyr Cys Trp Thr Trp Gly Leu Ala Cys Trp Cys
Glu Gly Leu Pro 35 40 45 Asp Asp Lys Thr Trp Lys Ser Glu Thr Asn
Thr Cys Gly 50 55 60 16460PRTLeiurus quinquestriatus hebraeus
164Asp Gly Tyr Ile Arg Gly Asp Gly Cys Lys Val Ser Cys Val Ile Asn
1 5 10 15 His Val Phe Cys Asp Asn Glu Cys Lys Ala Ala Gly Gly Ser
Tyr Gly 20 25 30 Tyr Cys Trp Ala Trp Gly Leu Ala Cys Trp Cys Glu
Gly Leu Pro Ala 35 40 45 Glu Arg Glu Trp Lys Tyr Glu Thr Asn Thr
Cys Gly 50 55 60 16562PRTButhotus judaicus 165Asp Gly Tyr Ile Arg
Lys Lys Asp Gly Cys Lys Val Ser Cys Ile Ile 1 5 10 15 Gly Asn Glu
Gly Cys Arg Lys Glu Cys Val Ala His Gly Gly Ser Phe 20 25 30 Gly
Tyr Cys Trp Thr Trp Gly Leu Ala Cys Trp Cys Glu Asn Leu Pro 35 40
45 Asp Ala Val Thr Trp Lys Ser Ser Thr Asn Thr Cys Gly Arg 50 55 60
16661PRTButhacus arenicola 166Asp Gly Tyr Ile Arg Arg Arg Asp Gly
Cys Lys Val Ser Cys Leu Phe 1 5 10 15 Gly Asn Glu Gly Cys Asp Lys
Glu Cys Lys Ala Tyr Gly Gly Ser Tyr 20 25 30 Gly Tyr Cys Trp Thr
Trp Gly Leu Ala Cys Trp Cys Glu Gly Leu Pro 35 40 45 Asp Asp Lys
Thr Trp Lys Ser Glu Thr Asn Thr Cys Gly 50 55 60 16761PRTLeiurus
quinquestriatus quinquestriatus 167Asp Gly Tyr Ile Arg Lys Arg Asp
Gly Cys Lys Leu Ser Cys Leu Phe 1 5 10 15 Gly Asn Glu Gly Cys Asn
Lys Glu Cys Lys Ser Tyr Gly Gly Ser Tyr 20 25 30 Gly Tyr Cys Trp
Thr Trp Gly Leu Ala Cys Trp Cys Glu Gly Leu Pro 35 40 45 Asp Asp
Lys Thr Trp Lys Ser Glu Thr Asn Thr Cys Gly 50 55 60 16860PRTBothus
occitanus tunetanus 168Asp Gly Tyr Ile Lys Gly Tyr Lys Gly Cys Lys
Ile Thr Cys Val Ile 1 5 10 15 Asn Asp Asp Tyr Cys Asp Thr Glu Cys
Lys Ala Glu Gly Gly Thr Tyr 20 25 30 Gly Tyr Cys Trp Lys Trp Gly
Leu Ala Cys Trp Cys Glu Asp Leu Pro 35 40 45 Asp Glu Lys Arg Trp
Lys Ser Glu Thr Asn Thr Cys 50 55 60 16963PRTLeiurus
quinquestriatus hebraeus 169Asp Asn Gly Tyr Leu Leu Asn Lys Ala Thr
Gly Cys Lys Val Trp Cys 1 5 10 15 Val Ile Asn Asn Ala Ser Cys Asn
Ser Glu Cys Lys Leu Arg Arg Gly 20 25 30 Asn Tyr Gly Tyr Cys Tyr
Phe Trp Lys Leu Ala Cys Tyr Cys Glu Gly 35 40 45 Ala Pro Lys Ser
Glu Leu Trp Ala Tyr Ala Thr Asn Lys Cys Asn 50 55 60 17062PRTTityus
serrulatus 170Lys Glu Gly Tyr Leu Met Asp His Glu Gly Cys Lys Leu
Ser Cys Phe 1 5 10 15 Ile Arg Pro Ser Gly Tyr Cys Gly Arg Glu Cys
Gly Ile Lys Lys Gly 20 25 30 Ser Ser Gly Tyr Cys Tyr Ala Trp Pro
Ala Cys Tyr Cys Tyr Gly Leu 35 40 45 Pro Asn Trp Val Lys Val Trp
Asp Arg Ala Thr Asn Lys Cys 50 55 60 17164PRTTityus zulianus 171Lys
Asp Gly Tyr Leu Val Gly Asn Asp Gly Cys Lys Tyr Ser Cys Phe 1 5 10
15 Thr Arg Pro Gly Thr Tyr Cys Ala Asn Glu Cys Ser Arg Val Lys Gly
20 25 30 Lys Asp Gly Tyr Cys Tyr Ala Trp Met Ala Cys Tyr Cys Tyr
Ser Met 35 40 45 Pro Asn Trp Val Lys Thr Trp Asp Arg Ala Thr Asn
Arg Cys Gly Arg 50 55 60 17229PRTOldenlandia affinis 172Gly Leu Pro
Val Cys Gly Glu Thr Cys Val Gly Gly Thr Cys Asn Thr 1 5 10 15 Pro
Gly Cys Thr Cys Ser Trp Pro Val Cys Thr Arg Asn 20 25
17329PRTOldenlandia affinis 173Cys Gly Glu Thr Cys Phe Gly Gly Thr
Cys Asn Thr Pro Gly Cys Ser 1 5 10 15 Cys Thr Trp Pro Ile Cys Thr
Arg Asp Gly Leu Pro Val 20 25 17430PRTOldenlandia affinis 174Gly
Thr Pro Cys Gly Glu Ser Cys Val Tyr Ile Pro Cys Ile Ser Gly 1 5 10
15 Val Ile Gly Cys Ser Cys Thr Asp Lys Val Cys Tyr Leu Asn 20 25
30
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