U.S. patent application number 10/386529 was filed with the patent office on 2003-09-25 for antifreeze proteins from basidiomycetes.
This patent application is currently assigned to NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND TECHNOLOGY. Invention is credited to Hoshino, Tamotsu, Kiriaki, Michiko, Kondo, Hidemasa, Ohgiya, Satoru, Tsuda, Sakae, Yokota, Yuji, Yumoto, Isao.
Application Number | 20030180884 10/386529 |
Document ID | / |
Family ID | 27767242 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030180884 |
Kind Code |
A1 |
Hoshino, Tamotsu ; et
al. |
September 25, 2003 |
Antifreeze proteins from basidiomycetes
Abstract
The present invention provides antifreeze proteins produced by a
basidiomycete. The antifreeze protein has a high antifreeze
activity such as a thermal hysteresis activity or an
ice-recrystallization inhibition activity.
Inventors: |
Hoshino, Tamotsu; (Hokkaido,
JP) ; Kiriaki, Michiko; (Hokkaido, JP) ;
Tsuda, Sakae; (Hokkaido, JP) ; Ohgiya, Satoru;
(Hokkaido, JP) ; Kondo, Hidemasa; (Hokkaido,
JP) ; Yokota, Yuji; (Hokkaido, JP) ; Yumoto,
Isao; (Hokkaido, JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
WASHINGTON
DC
20037
US
|
Assignee: |
NATIONAL INSTITUTE OF ADVANCED
INDUSTRIAL SCIENCE AND TECHNOLOGY
|
Family ID: |
27767242 |
Appl. No.: |
10/386529 |
Filed: |
March 13, 2003 |
Current U.S.
Class: |
435/69.1 ;
252/71; 435/254.1; 435/320.1; 530/326; 536/23.7 |
Current CPC
Class: |
C07K 14/375
20130101 |
Class at
Publication: |
435/69.1 ;
530/326; 435/320.1; 435/254.1; 536/23.7; 252/71 |
International
Class: |
C07K 007/08; C07H
021/04; C09K 005/00; C12N 001/14; C12P 021/02 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 15, 2002 |
JP |
2002-072612 |
Mar 5, 2003 |
JP |
2003-57888 |
Claims
What is claimed is:
1. An isolated antifreeze protein produced by a basidiomycete.
2. The isolated antifreeze protein as defined in claim 1, wherein
said basidiomycete is a member of the order Aphyllophorales.
3. The isolated antifreeze protein as defined in claim 2, wherein
said basidiomycete is a member of the family Ramariaceae.
4. The isolated antifreeze protein as defined in claim 1, wherein
said basidiomycete is a member of the order Agaricales.
5. The isolated antifreeze protein as defined in claim 4, wherein
said basidiomycete is a member of the family Coprinaceae or
Tricholomataceae.
6. The isolated antifreeze protein as defined in any one of claims
1 to 5, which depresses the freezing point of an aqueous
solution.
7. The isolated antifreeze protein as defined in claim 1, wherein
said protein comprises an N-terminal amino acid sequence selected
from the group consisting of:
Ala-Gly-Pro-Ser-Ala-Val-Ala-Gly-Leu-Thr-Ala-Gly-Asn--
Tyr-Ala-Ile-Leu-Ala-Ser-Thr (SEQ ID NO: 1),
Ala-Gly-Pro-Ser-Ala-Val-Pro-Le-
u-Gly-Thr-Ala-Gly-Asn-Tyr-Val-Ile-Leu-Ala-Ser-Thr (SEQ ID NO: 2),
Ala-Gly-Pro-Thr-Ala-Val-Pro-Leu-Gly-Thr-Ala-Gly-Asn-Tyr-Ala-Ile-Leu-Ala-S-
er-Thr (SEQ ID NO: 3),
Ala-Gly-Pro-Ser-Ala-Val-Pro-Leu-Gly-Thr-Ala-Gly-Asn-
-Tyr-Ala-Ile-Leu-Ala-Ser-Thr (SEQ ID NO: 4),
Ala-Gly-Pro-Thr-Ala-Val-Asn-L-
eu-Gly-Thr-Ala-Lys-Asn-Tyr-Ala-Ile-Leu-Thr-Lys-Ala (SEQ ID NO: 5),
Ala-Gly-Pro-Thr-Ala-Val-Asn-Leu-Gly-Thr-Ala-Lys-Thr-Tyr-Ala-Ile-Leu-Thr-L-
ys-Ala (SEQ ID NO: 6),
Ala-Gly-Pro-Thr-Ala-Val-Asn-Leu-Gly-Thr-Ala-Lys-Asn-
-Tyr-Ala-Ile-Leu-Thr-Lys-Thr (SEQ ID NO: 7), and an N-terminal
amino acid sequence substantially homologous to any one of SEQ ID
NOS: 1 to 7.
8. An isolated polypeptide comprising an N-terminal amino acid
sequence selected from the group consisting of:
Ala-Gly-Pro-Ser-Ala-Val-Ala-Gly-Le-
u-Thr-Ala-Gly-Asn-Tyr-Ala-Ile-Leu-Ala-Ser-Thr (SEQ ID NO: 1),
Ala-Gly-Pro-Ser-Ala-Val-Pro-Leu-Gly-Thr-Ala-Gly-Asn-Tyr-Val-Ile-Leu-Ala-S-
er-Thr (SEQ ID NO: 2),
Ala-Gly-Pro-Thr-Ala-Val-Pro-Leu-Gly-Thr-Ala-Gly-Asn-
-Tyr-Ala-Ile-Leu-Ala-Ser-Thr (SEQ ID NO: 3),
Ala-Gly-Pro-Ser-Ala-Val-Pro-L-
eu-Gly-Thr-Ala-Gly-Asn-Tyr-Ala-Ile-Leu-Ala-Ser-Thr (SEQ ID NO: 4),
Ala-Gly-Pro-Thr-Ala-Val-Asn-Leu-Gly-Thr-Ala-Lys-Asn-Tyr-Ala-Ile-Leu-Thr-L-
ys-Ala (SEQ ID NO: 5),
Ala-Gly-Pro-Thr-Ala-Val-Asn-Leu-Gly-Thr-Ala-Lys-Thr-
-Tyr-Ala-Ile-Leu-Thr-Lys-Ala (SEQ ID NO: 6),
Ala-Gly-Pro-Thr-Ala-Val-Asn-L-
eu-Gly-Thr-Ala-Lys-Asn-Tyr-Ala-Ile-Leu-Thr-Lys-Thr (SEQ ID NO: 7),
and an N-terminal amino acid sequence substantially homologous to
any one of SEQ ID NOS: 1 to 7, wherein said polypeptide has a
molecular mass of 15 to 30 kDa, and depresses the freezing point of
an aqueous solution.
9. An isolated polypeptide comprising a polypeptide selected from
the group consisting of an amino acid sequence shown in any one of
SEQ ID NOS: 9, 11, 13, 15, 17, 19 and 21, and an amino acid
sequence having one or more amino acid deletions, substitutions or
additions relative to an amino acid sequence shown in any one of
SEQ ID NOS: 9, 11, 13, 15, 17, 19 and 21, wherein said polypeptide
depresses the freezing point of an aqueous solution.
10. An isolated polynucleotide encoding a polypeptide selected from
the group consisting of an amino acid sequence shown in any one of
SEQ ID NOS: 9, 11, 13, 15, 17, 19 and 21, and an amino acid
sequence having one or more amino acid deletions, substitutions or
additions relative to an amino acid sequence shown in any one of
SEQ ID NOS: 9, 11, 13, 15, 17, 19 and 21, wherein said polypeptide
depresses the freezing point of an aqueous solution.
11. An isolated polynucleotide comprising a polynucleotide selected
from the group consisting of a polynucleotide shown in any one of
SEQ ID NOS: 8, 10, 12, 14, 16, 18 and 20, and a polynucleotide
which hybridizes under stringent conditions with a polynucleotide
complementary to a polynucleotide consisting of all or a part of a
base sequence shown in any one of SEQ ID NOS: 8, 10, 12, 14, 16, 18
and 20, wherein said polynucleotide encodes a protein which
depresses the freezing point of an aqueous solution.
12. A recombinant vector comprising a polynucleotide as defined in
claim 10 or 11.
13. A transformant comprising the recombinant vector as defined in
claim 12.
14. A method of preparing an antifreeze protein, comprising
culturing the transformant as defined in claim 13 under conditions
such that said transformant produces said antifreeze protein, and
collecting the antifreeze protein produced from the resulting
culture.
15. An antifreezing composition comprising the protein as defined
in claim 1 or the polypeptide as defined in claim 9, and a carrier
or diluent.
16. An antibody which reacts specifically with the protein as
defined in claim 1 or the polypeptide of claim 9.
17. A protein-antibody complex, said complex comprising a protein
bound by an antibody that specifically recognizes and binds an
epitope of said protein, wherein said antibody is an antibody as
defined in claim 16, and wherein said complex depresses the
freezing point of an aqueous solution.
18. A method of preparing an antifreeze protein, comprising
culturing a basidiomycete that produces an antifreeze protein under
low temperature and conditions such that said basidiomycete
produces said antifreeze protein, and collecting the produced
antifreeze produced from the resulting culture.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel proteins originated
from fungi. In particular, the present invention relates to
antifreeze proteins excellent in antifreeze activity and useable as
ice recrystallization inhibitors and cryopreservatives, and a
method of preparing the same.
BACKGROUND OF THE INVENTION
[0002] A protein exhibiting an antifreeze effect on aqueous
solutions is generally referred to as an antifreeze protein (AFP).
Various antifreeze protein have been found in living organisms such
as fish, insects, plants, fungi and bacteria which typically have
adaptability to low temperature environments. It is know that all
antifreeze proteins originated from fish and plants allow an ice
nucleus to grow to an ice crystal having a bi-pyramid shape, just
like a pair of triangular pyramids joined together at their bottom
surfaces. This mechanism is explained as follows. Under usual
conditions, upon generation of an ice nucleus in an aqueous
solution, an ice crystal first grows to have a flat hexagonal plate
shape. In this case, the ice crystal has a growth rate in the
vertical direction 100 times lower than that in the plate plane
direction. In contrast, when an antifreeze protein is present in
the aqueous solution, the ice crystal gradually grows to the
bi-pyramid-shaped ice crystal under restraint on its growth in the
plate plane direction in such manner that a plate-shaped body is
initially formed to provide a base plane, and a plurality of small
plate-shaped bodies are sequentially piled up on both sides of the
plate-shaped body in the vertical direction with respect to the
base plane.
[0003] An antifreeze protein dissolved in an aqueous solution
brings about an antifreeze effect on the aqueous solution, such as
1) thermal hysteresis, 2) ice-recrystallization inhibition, and 3)
ice crystal shape control. While the water freezing point is
generally equal to the ice melting point, an aqueous solution
containing an antifreeze protein has a depressed water freezing
point because the protein is bonded to an ice crystal to be formed.
This phenomenon is referred to as "thermal hysteresis," and the
difference between the melting point of the ice formed therein and
the water freezing point is defined as "depression of freezing
point." A greater depression of freezing point means a greater
antifreeze effect. The ice crystal formed therein grows while
absorbing water generated by sublimation or partial melting at a
relatively high temperature of -10.degree. C. or more. Inhibition
of this phenomenon is defined as "ice-recrystallization
inhibition." A higher ice-recrystallization-inhibition activity
means a higher antifreeze effect. By taking advantage of the above
properties of the antifreeze protein, the antifreeze protein has
been proposed for use as an additive for ice cream apt to
deteriorate in its flavor or taste due to
attachment/recrystallization of water molecules in ambient air
caused by cold insulation, or as a cryopreservative for cells and
organs. The antifreeze protein is also expected to function as an
effective additive for eliminating clogging of pipelines due to ice
recrystallization in a system using ice slurry, such as cryogenic
supply systems or cryogenic storage systems.
[0004] However, it is difficult to assure a large, stable supply of
most of the known antifreeze proteins originated from plants and
animals. Therefore, recombinant gene technology has been used to
produce some antifreeze proteins originated from fish or insects,
and to make the proteins more stable. However, antifreeze proteins
produced by recombinant methods have not been used in foods for
human consumption because of consumer opposition to gene-altered
products. While some antifreeze proteins have been successfully
purified from bacteria, they are not suitable for human consumption
due to the properties related to their bacterial origin, and due to
insufficient stability. While it has been reported that some
antifreeze proteins exist in basidiomycetes, which are widely
utilized for human consumption, no antifreeze protein has been
isolated/purified therefrom.
[0005] While various attempts have heretofore been made to put
natural antifreeze proteins mainly originated from plants or fish
to practical use as a quality improving agent for frozen foods such
as ice cream, as a cryopreservative for cells, and as an additive
for cryogenic supply systems or cryogenic storage systems, no
practical application has been achieved due to instability in
activity of the conventional antifreeze proteins and the resulting
need for using them in large quantities to bring about desired
functions in view of their poor stability.
SUMMARY OF THE INVENTION
[0006] It is therefore an object of the present invention to
provide an antifreeze protein having a high antifreeze activity,
and capable of being preparing in large quantities at a low
cost.
[0007] In order to achieve this object, through various research
the present inventors have found that basidiomycetes, such as
Typhula ishikariensis, produce novel proteins having a high
antifreeze activity, and that antifreeze proteins meeting the above
objects can be isolated and purified therefrom. Based on this
knowledge, the inventors have accomplished the present invention
relating to novel proteins which are produced by basidiomycetes and
secreted out of cells thereof, and a method of preparing the
antifreeze proteins.
[0008] Specifically, according to a first aspect of the present
invention, there is provided antifreeze proteins which are produced
by basidiomycetes.
[0009] In the first aspect of the present invention, the
basidiomycetes may be a fungi belonging to the order
Aphyllophorales. The fungi belonging to the order Aphyllophorales
may be a fungi belonging to the family Ramariaceae.
[0010] Alternatively, the basidiomycetes may be a fungi belonging
to the order Agaricales. The fungi belonging to the order
Agaricales may belong to the family Coprinaceae or the family
Tricholomataceae.
[0011] Preferably, the basidiomycetes is Typhula ishikariensis.
[0012] The antifreeze proteins according to the first aspect of the
present invention have the ability to depress the freezing point of
an aqueous solution.
[0013] The antifreeze proteins according to the first aspect of the
present invention have an N-terminal amino acid sequence selected
from the group consisting of:
[0014]
Ala-Gly-Pro-Ser-Ala-Val-Ala-Gly-Leu-Thr-Ala-Gly-Asn-Tyr-Ala-Ile-Leu-
-Ala-Ser-Thr (SEQ ID NO: 1),
[0015]
Ala-Gly-Pro-Ser-Ala-Val-Pro-Leu-Gly-Thr-Ala-Gly-Asn-Tyr-Val-Ile-Leu-
-Ala-Ser-Thr (SEQ ID NO: 2),
[0016]
Ala-Gly-Pro-Thr-Ala-Val-Pro-Leu-Gly-Thr-Ala-Gly-Asn-Tyr-Ala-Ile-Leu-
-Ala-Ser-Thr (SEQ ID NO: 3),
[0017]
Ala-Gly-Pro-Ser-Ala-Val-Pro-Leu-Gly-Thr-Ala-Gly-Asn-Tyr-Ala-Ile-Leu-
-Ala-Ser-Thr (SEQ ID NO: 4),
[0018]
Ala-Gly-Pro-Thr-Ala-Val-Asn-Leu-Gly-Thr-Ala-Lys-Asn-Tyr-Ala-Ile-Leu-
-Thr-Lys-Ala (SEQ ID NO: 5),
[0019]
Ala-Gly-Pro-Thr-Ala-Val-Asn-Leu-Gly-Thr-Ala-Lys-Thr-Tyr-Ala-Ile-Leu-
-Thr-Lys-Ala (SEQ ID NO: 6),
[0020]
Ala-Gly-Pro-Thr-Ala-Val-Asn-Leu-Gly-Thr-Ala-Lys-Asn-Tyr-Ala-Ile-Leu-
-Thr-Lys-Thr (SEQ ID NO: 7), and
[0021] an N-terminal amino acid sequence substantially homologous
to any one of SEQ ID NOS: 1 to 7.
[0022] According to a second aspect of the present invention, there
is provided a polypeptide comprising an N-terminal amino acid
sequence selected from the group consisting of:
[0023]
Ala-Gly-Pro-Ser-Ala-Val-Ala-Gly-Leu-Thr-Ala-Gly-Asn-Tyr-Ala-Ile-Leu-
-Ala-Ser-Thr (SEQ ID NO: 1),
[0024]
Ala-Gly-Pro-Ser-Ala-Val-Pro-Leu-Gly-Thr-Ala-Gly-Asn-Tyr-Val-Ile-Leu-
-Ala-Ser-Thr (SEQ ID NO: 2),
[0025]
Ala-Gly-Pro-Thr-Ala-Val-Pro-Leu-Gly-Thr-Ala-Gly-Asn-Tyr-Ala-Ile-Leu-
-Ala-Ser-Thr (SEQ ID NO: 3),
[0026]
Ala-Gly-Pro-Ser-Ala-Val-Pro-Leu-Gly-Thr-Ala-Gly-Asn-Tyr-Ala-Ile-Leu-
-Ala-Ser-Thr (SEQ ID NO: 4),
[0027]
Ala-Gly-Pro-Thr-Ala-Val-Asn-Leu-Gly-Thr-Ala-Lys-Asn-Tyr-Ala-Ile-Leu-
-Thr-Lys-Ala (SEQ ID NO: 5),
[0028]
Ala-Gly-Pro-Thr-Ala-Val-Asn-Leu-Gly-Thr-Ala-Lys-Thr-Tyr-Ala-Ile-Leu-
-Thr-Lys-Ala (SEQ ID NO: 6),
[0029]
Ala-Gly-Pro-Thr-Ala-Val-Asn-Leu-Gly-Thr-Ala-Lys-Asn-Tyr-Ala-Ile-Leu-
-Thr-Lys-Thr (SEQ ID NO: 7), and
[0030] an N-terminal amino acid sequence substantially homologous
to any one of SEQ ID NOS: 1 to 7. This polypeptide has a molecular
mass of 15 to 30 kDa, and the ability to depress the freezing point
of an aqueous solution.
[0031] According to a third aspect of the present invention, there
is provided a polypeptide comprising the following amino acid
sequence (a) or (b):
[0032] an amino acid sequence shown in any one of SEQ ID NOS: 9,
11, 13, 15, 17, 19 and 21;
[0033] an amino acid sequence having one or more amino acid
deletions, substitutions or additions relative to an amino acid
sequence shown in any one of SEQ ID NOS: 9, 11, 13, 15, 17, 19 and
21. This polypeptide has the ability to depress the freezing point
of an aqueous solution.
[0034] According to a fourth aspect of the present invention, there
is provided a polynucleotide encoding the following polypeptide (a)
or (b):
[0035] a polypeptide comprising an amino acid sequence shown in any
one of SEQ ID NOS: 9, 11, 13, 15, 17, 19 and 21;
[0036] a polypeptide comprising an amino acid sequence having one
or more amino acid deletions, substitutions or additions relative
to an amino acid sequence shown in any one of SEQ ID NOS: 9, 11,
13, 15, 17, 19 and 21. This polypeptide (a) or (b) has the ability
to depress the freezing point of an aqueous solution.
[0037] According to a fifth aspect of the present invention, there
is provided a polynucleotide comprising the following
polynucleotide sequence (a) or (b):
[0038] a polynucleotide comprising a base sequence shown in any one
of SEQ ID NOS: 8, 10, 12, 14, 16, 18 and 20;
[0039] a polynucleotide which hybridizes under stringent conditions
with a polynucleotide comprising a base sequence complementary to a
polynucleotide consisting of all or a part of a base sequence shown
in any one of SEQ ID NOS: 8, 10, 12, 14, 16, 18 and 20. This
polynucleotide (a) or (b) encodes a polypeptide which has the
ability to depress the freezing point of an aqueous solution.
[0040] According to a six aspect of the present invention, there is
provided a recombinant vector containing the polynucleotide set
forth in the fourth or fifth aspect of the present invention.
[0041] According to a seventh aspect of the present invention,
there is provided a transformant containing the recombinant vector
set forth in the sixth aspect of the present invention.
[0042] According to an eighth aspect of the present invention,
there is provided a method of preparing an antifreeze protein,
which comprises culturing the transformant set forth in the seventh
aspect of the present invention, and collecting the antifreeze
protein from the resulting culture.
[0043] According to a ninth aspect of the present invention, there
is provided an antifreezing agent containing the protein set forth
in any one of the first to third aspects of the present
invention.
[0044] According to a tenth aspect of the present invention, there
is provided an antibody which reacts specifically with the protein
set forth in any one of the first to third aspects of the present
invention.
[0045] According to an eleventh aspect of the present invention,
there is provided a polypeptide-antibody complex comprising a
polypeptide and an antibody bound to the polypeptide through an
immune reaction. The antibody specifically recognizes and binds to
an epitope of a protein or polypeptide set forth in any one of the
first to third aspects of the present invention. The
polypeptide-antibody complex has the ability to depress the
freezing point of an aqueous solution.
[0046] According to a twelfth aspect of the present invention,
there is provided a method of preparing an antifreeze protein,
which comprises culturing a basidiomycete capable of producing the
antifreeze protein, under low temperature, and collecting the
produced antifreeze protein from the resulting cultured
solution.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIG. 1 includes pictures of respective ice crystals formed
in a sample containing an antifreeze protein originated from fish
(Type III) (A), a sample containing an antifreeze protein
originated from Typhula ishikariensis (B), a Pleurotus ostreatus
culture solution (C) and a Flammulina velutipes culture solution
(D), each subjected to a low temperature treatment., An ice crystal
having a typical bi-pyramidal structure is formed in the sample
containing the antifreeze protein originated from fish, and ice
crystals having different indented shapes, specifically a
chipped-stone-tool shape, and a star shape, are formed in the
samples containing the antifreeze protein originated from Typhula
ishikariensis and the Flammulina velutipes culture solution,
respectively. A spherical ice crystal is formed in the Pleurotus
ostreatus culture solution.
[0048] FIG. 2 is a graphical representation of the freezing point
depressions in respective solutions containing the antifreeze
protein originated from Typhula ishikariensis and the antifreeze
protein originated from fish (Type III).
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0049] The term "antifreeze protein" herein has an ordinary meaning
commonly recognized in the art, and means a protein having an
activity that inhibits ice-crystal growth.
[0050] An antifreeze protein according to one embodiment of the
present invention is contained in an extract from basidiomycetes. A
basidiomycete to be used in the present invention may be any strain
capable of producing a protein having an antifreeze activity,
preferably a strain capable of growing at a low temperature of
4.degree. C. or less.
[0051] Preferably, the basidiomycetes belong to Aphyllophorales or
Agaricales. The basidiomycetes belonging to Aphyllophorales include
fungi belonging to Cantharellaceae, Polyporaceae, Ganodermataceae,
Hydnaceae, Schizophyllaceae, Coniophoraceae, Ramariaceae,
Stereaceae, and Thelephoraceae. The basidiomycetes belonging to
Agaricales include fungi belonging to Hygrophoraceae,
Tricholomataceae, Amanitaceae, Agaricaceae, Coprinaceae,
Strophariaceae, Cortinariaceae, Boletaceae, and Russulaceae.
[0052] Preferably, the basidiomycetes include: fungi belonging to
Aphyllophorales-Ramariaceae-Typhula, such as Typhula ishikariensis,
T. incarnata or T. phacorrhiza; fungi belonging to
Agaricales-Coprinaceae-Co- prinus (Coprinus psychromorbidus); and
fungi belonging to Agaricales-Tricholomataceae-Flammulina, such as
Flammulina velutipes. More preferably, the basidiomycetes are fungi
belonging to Typhula and Coprinus. The Typhula ishikariensis BRB
strain and the Coprinus psychromorbidus CCFC006721 strain may be
used as particularly preferable basidiomycetes.
[0053] The Typhula ishikariensis BRB strain is a new strain
isolated from the natural environment. The Typhula ishikariensis
BRB strain exhibits the following microbiological properties.
[0054] One or several fruit-bodies each having a length of 0.5 cm
are generated from each of the sclerotia. The fruit body includes a
cylindrical club-shaped top portion having a length of 0.2-0.5 cm
(about 3 cm under artificial conditions), a diameter of 0.5-2.0 mm,
and a color of white or approximate-white which will change to
light champagne after maturation. Thus, the top portion is
obviously distinguishable from the stem portion of the fruit body.
A basidium has a club shape and bears 4 spores. Each of the spores
has a flat shape of about 10.times.5 .mu.m. The sclerotium has a
spherical, oval or indefinite shape having a diameter of 0.5-3 mm,
and a dark-brown wet body which will change to black when dried. In
view of the above microbiological properties, the above strain can
be classified into Typhula ishikariensis on the basis of
"Nihon-kingakkaiho," Vol. 2, Basidiomycetes No. 4" as a references
of classification/identification. This strain "Typhula
ishikariensis BRB" was deposited as deposit number FERM P-18741 in
International Patent Organism Depositary, National Institute of
Advanced Industrial Science and Technology, Tsukuba Central 6,
1-1-1 Higashi, Tsukuba, Ibaraki, Japan (zip code: 305-5466) on Feb.
27, 2002.
[0055] The Coprinus psychromorbidus CCFC006721 strain was deposited
as deposit number CCFC006721 in the Canadian Collection of Fungal
Cultures, Agriculture and Agri-Food Canada, Rm. 1015, K. W. Neatby
Bldg., Ottawa, Ontario, K1A 0C6 CANADA.
[0056] The antifreeze proteins of the present invention can be
prepared by culturing a specific basidiomycete in a culture medium
and collecting an antifreeze protein from the resulting culture
solution. The culture medium to be used for culturing the
basidiomycete is not limited to a specific form, but any suitable
natural or synthetic culture medium containing an appropriate
amount of nutritional elements required for activating the strain,
such as carbon source, nitrogen source or inorganic substance may
be used. For example, the medium may include a potato-dextrose
medium and a cornmeal medium which are typically used for culturing
filamentous fungi. The carbon source for use in the synthetic
medium may include soluble starch, glucose and maltose. The
nitrogen source may include a nitrogen-containing natural product
such as peptones, yeast extracts or meat extracts, and a
nitrogen-containing inorganic compound such as sodium nitrate or
ammonium chloride. The inorganic substance may include potassium
phosphate, sodium phosphate, magnesium sulfate, calcium chloride
and ferric chloride. The culturing method typically includes, but
not limited to, a shaking culturing method, an aeration/agitation
culturing method and a two-step culturing method. The culturing
temperature is set at any low temperature, preferably in the range
of 0 to 15.degree. C., more preferably 0.degree. C. or less.
Alternatively, cells may be sufficiently proliferated at an optimal
growth temperature, and then transferred into another medium to
culture them at 0.degree. C. or less. A culturing period is
typically 1 to 7 weeks.
[0057] The antifreeze proteins of the present invention can be
purified through any conventional purification method commonly used
in the art. The cultivated cells may be separated from the culture
medium, for example, through centrifugation, filtration or
ultrafiltration. The antifreeze proteins contained in the
supernatant of a culture solution resulting from the separation of
the cells can be isolated/purified through a salting-out method
using ammonium sulfate or sodium sulfate, an organic-solvent
precipitation method using acetone or ethanol, a column
chromatography method using a cation exchanger (e.g. CM, S, SP) or
an anion exchanger (e.g. DEAE, Q, QAE), or a gel filtration method
using agarose derivatives.
[0058] The inventors also isolated antifreeze proteins, and a
polynucleotides encoding the proteins, from the strain "Typhula
ishikariensis BRB." The polynucleotides encoding the antifreeze
proteins can be isolated through any suitable method commonly used
in the art (see, for example, WO 00/188045).
[0059] The antifreeze proteins of the present invention may
comprise an amino acid sequence shown in any one of SEQ ID NOS: 9,
11, 13, 15, 17, 19 and 21, and the polynucleotides encoding the
antifreeze proteins of the present invention may comprise a base
sequence shown in any one of SEQ ID NOS: 8, 10, 12, 14, 16, 18 and
20. As long as the proteins comprising the above amino acid
sequences have the ability to depress the freezing point of an
aqueous solution, the amino acid sequences may include a variation
or mutation such as one or more amino acid deletions, substitutions
or additions.
[0060] Thus, the antifreeze proteins of the present invention
include those proteins substantially homologous to any one of the
amino acid sequences shown in SEQ ID NOS: 9, 11, 13, 15, 17, 19 and
21.
[0061] The term "substantially homologous" as used throughout means
that two polypeptides have at least 80%, preferably 90% or more,
more preferably 95 to 100%, common amino acids.
[0062] For example, in the amino acid sequences shown in any one of
SEQ ID NOS: 9, 11, 13, 15, 17, 19 and 21, one amino acid,
preferably 10 to 20 amino acids, more preferably 5 to 10 amino
acids, may be deleted therefrom or substituted with different amino
acids. Alternatively, or in addition, one amino acid, preferably 10
to 20 amino acids, more preferably 5 to 10 amino acids may be added
to the amino acid sequence shown in any one of SEQ ID NOS: 9, 11,
13, 15, 17, 19 and 21. The polynucleotides encoding the antifreeze
proteins of the present invention also include polynucleotides
which hybridizes under stringent conditions with a polynucleotide
comprising a base sequence complementary to a polynucleotide
consisting of all or a part of the base sequence shown in any one
of SEQ ID NOS: 8, 10, 12, 14, 16, 18 and 20, and encodes a protein
having the ability to depress the freezing point of an aqueous
solution. The term "stringent conditions" herein means conditions
under which a specific hybrid is formed without formation of a
non-specific hybrids, or conditions under which a polynucleotide
having a high homology (homology: 90% or more, preferably 95% or
more) to the polynucleotide encoding the antifreeze protein is
hybridized. More specifically, such conditions can be achieved by
performing hybridization at 42 to 68.degree. C. under the presence
of 0.5 to 1 M NaCl, and then rinsing a filter at room temperature
to 68.degree. C. by using a 0.1 to 2 times concentration of SSC
(saline sodium citrate) solution. Preferably, stringent conditions
mean hybridization at 68.degree. C. in the presence of 1 M NaCl,
and washing at 68.degree. C. in a 2.times. concentration of SSC
solution.
[0063] The term "part of the base sequence" herein means a base
sequence of a polynucleotide including a part of the base sequence
of the above polynucleotide encoding the antifreeze protein,
wherein the polynucleotide encodes a protein having the ability to
depress the freezing point of an aqueous solution. The "part of the
base sequence" has a length sufficient to be hybridized under
stringent conditions. For example, it is constructed by at least 10
bases, preferably at least 50 bases, more preferably 200 bases.
[0064] A mutation can be introduced in the polynucleotide through a
conventional technique, such as a Kunkel method or a Gapped duplex
method, or a method based on these methods, for example, by using a
mutation-introducing kit utilizing a site-specific mutation
inducing method (e.g. Mutan-K available from TAKARA, MUTAN-G
available from TAKARA) or LA PCR in vitro Mutagenesis series kits
available from TAKARA. A polynucleotide having a base sequence
produced through the above technique can be produced through a
chemical synthesis method or a PCR method using a chromosomal DNA
as a template, or by using a polynucleotide fragment having the
base sequence as a probe to obtain the polynucleotide of the
present invention.
[0065] The antifreeze proteins may also be obtained by preparing a
recombinant vector containing the polynucleotide encoding a
antifreeze protein of the present invention, and culturing a
transformant having the recombinant vector introduced therein. The
recombinant vector of the present invention can be obtained by
linking all or a part of a polynucleotide of the present invention
to a suitable vector. The transformant of the present invention can
be obtained by introducing the recombinant vector of the present
invention into a host to allow a polynucleotide of the present
invention to be expressed. The term "part of the polynucleotide"
herein means a part of a polynucleotide encoding a antifreeze
protein capable of expressing a antifreeze protein of the present
invention when it is introduced into a host.
[0066] The recombinant vector used in the present invention is not
limited to a specific type, but may be any suitable vector capable
of cloning in host cells, such as plasmid, shuttle vector, phage or
helper plasmid. If the vector itself has no clonability, a DNA
fragment capable of providing clonability when it is inserted into
the chromosome of a host may be used in combination therewith.
[0067] The plasmid may be, but is not limited to, a plasmid
originated from Escherichia coli (e.g. pBR322, pBR325, pUC118,
pUC119, pUC18, pUC19 or pBluescript), a plasmid originated from
Bacillus subtilis (e.g. pUB 110 or pTP5), and a plasmid originated
from yeast (e.g. Yeps such as Yep 13, or Ycps such as Ycp 50). The
phage may be, but is not limited to, .lambda. phage (Charon4A,
Charon21A, EMBL3, EMBL4, .lambda. gt 10, .lambda. gt 11, or
.lambda. gt ZAP). An animal virus vector such as retrovirus or
vaccinia virus, or an insect virus vector such as Baculovirus may
also be used in combination.
[0068] The host is not limited to a specific type, but may include:
bacteria belonging to the Ralstonia genus such as Ralstonia
eutropha; bacteria belonging to the Pseudomonas genus such as
Pseudomonas putido; bacteria belonging to the Bacillus genus such
as Bacillus subtilis; bacteria belonging to the Escherichia genus
such as Escherichia coli; yeast belonging to the Saccharomyces
genus such as Saccharomyces cerevisiae; yeast belonging to the
Candida genus such as Candida maltosa; animal cells such as COS
cells, CHO cells, mice L cells, rat GH3 or human FL cells; and
insect cells such as SF9 cells.
[0069] When a bacterium such as Escherichia coli is used as a host,
it is preferable that the recombinant vector can exist
independently in the host, and comprises a promoter, a
polynucleotide of the present invention and a transcription
termination sequence. The promoter may be any suitable type capable
of being expressed in the host, for example, a promoter originated
from Escherichia coli or phage, such as a trp promoter, Lac
promoter, PL promoter, PE promoter or T7 promoter. The method used
to introduce the recombinant vector into cells may be, but is not
limited to, a method using calcium ions [Current Protocols in
Molecular Biology, 1, 181 (1994)] or an electroporation method.
[0070] When yeast is used as a host, the expression vector may be
YEp13 or YCp50. In this case, a promoter may include a gal 1
prompter, gal 10 promoter, heat-shock protein promoter, and GAP
promoter. The method used to introduce the recombinant vector into
yeast may be, but is not limited to, an electroporation method, a
Spheroplast method [Proc. Natl. Sci. USA, 84, 192, 9-1933 (1978)]
or a lithium acetate method [J. Bacteriol., 153, 163-168
(1983)].
[0071] An antifreeze protein of the present invention is obtained
by culturing a transformant of the present invention in a culture
medium to produce and accumulate an antifreeze protein in the
resulting culture (culture cells or culture supernatant), and
collecting the antifreeze protein from the culture. A transformant
of the present invention is cultured in a culture medium through a
conventional method for culturing a host. A culture medium for
culturing the transformant obtained by using bacteria such as
Escherichia coli as the host includes a complete medium such as LB
medium, and a synthetic medium such as M9 medium. The transformant
is aerobically cultured at a temperature of 25 to 37.degree. C. for
1 to 72 hours to accumulate the antifreeze protein in the cells,
and the accumulated antifreeze protein is collected. During
culturing, the pH value in the culture medium is maintained at
about 7. The pH value is adjusted using an inorganic acid, organic
acid or alkaline solution. The collected antifreeze protein can be
purified in the same manner as that described above.
[0072] The roughly purified antifreeze proteins or the entirely
purified antifreeze proteins obtained through the above process may
be used in a liquid form by adding thereto a stabilizer such as
glycerol, sucrose, or ethylene glycol, or may be used in a powder
form by drying it, for example, through a spray drying method or a
freeze-drying method.
[0073] As described above, the antifreeze proteins of the present
invention can be collected from a culture solution obtained by
culturing a specific basidiomycete or a transformant comprising a
polynucleotide encoding a antifreeze protein. Thus, the
basidiomycete strain or the transformant can be readily cultured on
a large scale by using an inexpensive medium to prepare the desired
antifreeze protein in large quantities at a low cost.
[0074] The present invention also includes a polypeptide-antibody
complex comprising a polypeptide and an antibody bound to the
polypeptide through an immune reaction. The complex has the ability
to depress the freezing point of an aqueous solution. Such a
protein can be prepared by immunizing an animal with the antifreeze
protein of the present invention and collecting the resulting
antibody (see, for example, WO 00/188045).
[0075] The term "antifreeze activity" herein means an activity of
inhibiting ice-crystal growth. The antifreeze activity can be
determine, for example, by observing the growth process of ice
crystals in a solution containing an antifreeze protein, and the
shape of the ice crystals being formed, or by measuring the
freezing point depression of the solution with an osmometer using a
freezing point depression method. More specifically, the presence
of the antifreeze activity in a specific protein can be determine
by identifying through microscopic observations the fact that no
ice-crystal growth is generated in a solution containing the
specific protein, or that an ice crystal formed in the solution
containing the specific protein has an indented shape, for example,
a chipped-stone-tool shape or a star shape. The level of the
antifreeze activity of the protein can also be determined in
proportion to the freezing point depression in the solution.
[0076] With respect to a first sample containing an antifreeze
protein of the present invention originated from a specific
basidiomycete and a second sample containing a conventional
antifreeze protein originated from fish, the growth process of an
ice-crystal, the shape of the formed ice crystal, and the freezing
point depressions were experimentally determined in both the
samples. As a result, it was verified that the ice crystal shape of
an antifreeze protein of the present invention originating from the
basidiomycete was different from that of the conventional
high-activity-type antifreeze protein originated from fish, and the
solution obtained from the first sample had a freezing point
depression about 1.3 times greater than that obtained from the
second sample. That is, the results show that an antifreeze protein
of the present invention has an antifreeze activity about 1.3 times
greater than that of the conventional antifreeze protein originated
from fish.
[0077] A liquefied or powdered antifreeze protein obtained through
the method of the present invention is excellent in antifreeze
activity and productivity, and can be advantageously used as a
quality-improving agent for frozen foods, a cryopreservative for
cells, and an additive for cryogenic supply systems or cryogenic
storage systems. The proteins of the present invention can also be
used to prepare an antifreezing agent. In the present invention,
plural kinds of proteins can be prepared, and mixed them together
to form a complex of antifreeze proteins. The extract from a
basidiomycete capable of producing an antifreeze protein also has
an antifreeze activity usable directly for the above
applications.
[0078] All publications, patents and patent application cited
herein are incorporated herein by reference in their entirety.
EXAMPLE
[0079] While the present invention will be described in more detail
in conjunction with the following Example, the invention is not
limited thereto.
Example 1
Preparation of Antifreeze Proteins Originated from Typhula
ishikariensis
[0080] 1 L of Potato-Dextrose liquid medium (available from Difco)
was put in an Erlenmeyer flask having a volume of 3 L, and
subjected to autoclave sterilization at 121.degree. C. for 15
minutes. The Typhula ishikariensis BRB strain (Deposit No: FERM
P-18741) as a spawn was inoculated into the medium, and cultured at
-1.degree. C. for 1 month to obtain a culture solution. The culture
solution was centrifugalized, and the obtained supernatant was
dialyzed. Then, the dialyzed solution was fractionated through Q-
and S-Bio-Gel column chromatography to obtain five kinds of
purified protein samples. The obtained proteins had the following
properties.
[0081] Through dodecyl sodium sulfate-polyacrylamide gel
electrophoresis, all of the molecular masses of the proteins were
in the range of 15 to 30 kDa, more specifically about 22 kDa.
Through a gel filtration method and a dynamic light scattering
method, it was also verified that each of the proteins was a
monomer. Each of the N-terminal sequences of the proteins was
determined through an Edman method. The following four kinds of
sequences were determined:
1
Ala-Gly-Pro-Ser-Ala-Val-Ala-Gly-Leu-Thr-Ala-Gly-Asn-Tyr-Ala-Ile-L-
eu-Ala-Ser-Thr, (SEQ ID NO:1) Ala-Gly-Pro-Ser-Ala-Val-Pro-
-Leu-Gly-Thr-Ala-Gly-Asn-Tyr-Val-Ile-Leu-Ala-Ser-Thr, (SEQ ID NO:2)
Ala-Gly-Pro-Thr-Ala-Val-Pro-Leu-Gly-Thr-Ala-Gly-Asn-Tyr-Ala-Ile--
Leu-Ala-Ser-Thr, and (SEQ ID NO:3) Ala-Gly-Pro-Ser-Ala-Val-
-Pro-Leu-Gly-Thr-Ala-Gly-Asn-Tyr-Ala-Ile-Leu-Ala-Ser-Thr. (SEQ ID
NO:4)
[0082] By checking with the Protein Sequence Database, it was
verified that all of the proteins were novel. It is also intended
that any proteins comprising an N-terminal amino acid sequence
substantially homologous to either one of the above four kinds of
N-terminal amino acid sequences are encompassed within the scope of
the present invention. The term "substantially homologous" herein
means that two polypeptides have at least 80%, preferably 90% or
more, more preferably 95 to 100%, common amino acids. The
N-terminal amino acid sequence of the remaining one of the proteins
could not be determined through the Edman method, likely because it
includes some kind of modification.
Example 2
Preparation of Antifreeze Proteins Originated from Coprinus
psychromorbidus
[0083] 1 L of Potato-Dextrose liquid medium (available from Difco)
was put in an Erlenmeyer flask having a volume of 3 L, and
subjected to autoclave sterilization at 121.degree. C. for 15
minutes. The Coprinus psychromorbidus CCFC006721 strain as a spawn
was inoculated into the medium, and cultured at -1.degree. C. for 1
month to obtain a culture Attorney Docket No. Q73754 solution. The
culture solution was centrifugalized, and the obtained supernatant
was dialyzed. Then, the dialyzed solution was fractionated through
Q- and S-Bio-Gel column chromatography to obtain three kinds of
purified protein samples. The obtained proteins had the following
properties.
[0084] Through dodecyl sodium sulfate-polyacrylamide gel
electrophoresis, all of the molecular masses of the proteins were
in the range of 15 to 30 kDa, more specifically about 23 kDa.
Through a gel filtration method, it was also verified that each of
the proteins was a monomer. Each of the N-terminal sequences of the
proteins was determined through an Edman method. The following
three kinds of sequences were determined:
2
Ala-Gly-Pro-Thr-Ala-Val-Asn-Leu-Gly-Thr-Ala-Lys-Asn-Tyr-Ala-Ile-L-
eu-Thr-Lys-Ala; (SEQ ID NO:5) Ala-Gly-Pro-Thr-Ala-Val-Asn-
-Leu-Gly-Thr-Ala-Lys-Thr-Tyr-Ala-Ile-Leu-Thr-Lys-Ala; and (SEQ ID
NO:6) Ala-Gly-Pro-Thr-Ala-Val-Asn-Leu-Gly-Thr-Ala-Lys-Asn-Tyr-Ala--
Ile-Leu-Thr-Lys-Thr. (SEQ ID NO:7)
[0085] By checking with the Protein Sequence Database, it was
verified that all of the proteins were novel. It is also intended
that any proteins comprising an N-terminal amino acid sequence
substantially homologous to either one of the above three kinds of
N-terminal amino acid sequences are encompassed within the scope of
the present invention. The term "substantially homologous" herein
means that two polypeptides they have at least 80%, preferably 90%
or more, more preferably 95 to 100%, common amino acids.
Example 3
Preparation of anti-Typhula ishikariensis-Originated Antifreeze
Protein Antibodies
[0086] A solution having 10 mg of a Typhula
ishikariensis-originated antifreeze protein (selected from the
polypeptides of SEQ ID NOS: 1-3) dissolved therein was added to 1
ml of 1 mM sodium acetate buffer solution (pH 4.0), and the
obtained solution was stirred at room temperature for three hours
to prepare an antigen solution. The antigen solution was stirred in
a syringe together with Freund's complete adjuvant (FCA) to form an
emulsion, and a rabbit (Japanese white) was immunized with the
emulsion. Subsequently, the rabbit was immunized at two-week
intervals four times total. On and after the 2nd immunization,
Freund's incomplete adjuvant was used instead of the FCA. A small
amount of blood sample was taken from the immunized rabbit, and the
increase of antibody value (500 ELISA-units/ml) was confirmed
through a dot plot analysis using purified antifreeze protein from
Typhula ishikariensis. Then, a large blood sample was taken from
the rabbit. The blood sample was left at room temperature for 3.5
hours, and then left in a frigidarium for 48 hours to form a blood
clot. Then, the blood clot was centrifuged at 3000 g to obtain
blood serum. The obtained serum was stored at 4.degree. C., and
used as the source of the antibody.
[0087] Culture solutions of different basidiomycetes species were
cultured at -1.degree. C. for one month and were used as samples
for checking the specificity of the immune reaction using the
antibody. Each of the samples were individually subjected to
dodecyl sodium sulfate-polyacrylamide gel electrophoresis, and then
the ability of the antifreeze protein in the individual samples to
be recognized by the antibody was checked though protein staining
and western blotting. As a result, it was verified that antifreeze
proteins in samples originated from T. incarnata, T. phacorrhiza,
Coprinus psychromorbidus, and Flammulina velutipes could each be
detected by the antibody produced using the Typhula ishikariensis
antifreeze protein as an immunogen.
Example 4
Measurement of Antifreeze Activity
[0088] 1. Observation of Ice-Crystal Growth
[0089] Both a conventional fish-originated antifreeze protein (Type
III) and the Typhula ishikariensis BRB strain-originated antifreeze
protein prepared in Example 1 (corresponding to SEQ ID NO:3) were
dissolved at a concentration of 0.25 mg/ml in 0.1 M ammonium
hydrogencarbonate buffer solution (pH 7.9) to form corresponding
samples. 3 .mu.l of each of the samples were placed on a first
cover glass of 1.6 mm diameter having a washer of 1.2 mm diameter
and 0.8 mm thickness attached thereto with manicure, and then a
second cover glass of 1.25 mm diameter was attached to the washer
with manicure to cover the first cover glass. This measuring cell
was placed on a refrigerating stage mounted on a microscope, and
covered with a cover slip. The refrigerating stage was connected to
a refrigerating system, and the temperature of the stage was
controlled by a controller of the refrigerating system. The sample
in the measuring cell was chilled down to -25.degree. C.
(-10.degree. C./minute), and frozen. Then, the sample was heated to
melt the ice formed there within, leaving only one ice nucleus. The
sample was gradually chilled (-0.05.degree. C./minute), and the
ice-crystal growth in the sample was recorded by a highly-sensitive
CCD camera system and a video recorder (see FIG. 1). While a
bi-pyramidal-shaped ice crystal was formed in the sample containing
the fish-originated antifreeze protein, a chipped-stone-tool-shaped
ice crystal was formed in the sample containing the Typhula
ishikariensis-originated antifreeze protein.
[0090] In observations of the Coprinus psychromorbidus-originated
antifreeze protein obtained in EXAMPLE 2 (corresponding to SEQ ID
NO:7), the formation of chipped-stone-tool-shaped ice crystal was
also confirmed. Further, respective samples of a Flammulina
velutipes culture solution and a Pleurotus ostreatus culture
solution each subjected to a low temperature treatment were
observed in the same way. As a result, it was verified that a
star-shaped ice crystal was formed in the Flammulina velutipes
culture solution, and a spherical ice crystal was formed in the
Pleurotus ostreatus culture solution.
[0091] No ice-crystal growth was observed in the samples containing
the Typhula ishikariensis-originated antifreeze protein, the
Coprinus psychromorbidus-originated antifreeze protein, and the
Flammulina velutipes culture solution, even after they were kept at
-5.degree. C. for 1 hour. In contrast, a specific ice-crystal
growth was observed in the sample of the Pleurotus ostreatus
culture solution.
[0092] 2. Measurement of Freezing Point
[0093] Both a fish-originated antifreeze protein (Type III) and the
Typhula ishikariensis strain-originated antifreeze protein prepared
in Example 1 (corresponding to SEQ ID NO:3) was dissolved at
various concentrations in 0.1 M ammonium hydrogencarbonate buffer
solution (pH 7.9) to form corresponding samples. With respect to 50
.mu.l of each of the samples, a total osmotic pressure was measured
with an osmometer through a freezing point depression method, and a
freezing point was calculated in accordance with the measured total
osmotic pressure. In a low concentration range, the sample
containing the Typhula ishikariensis-originated antifreeze protein
is exhibited approximately the same freezing point as that of the
sample containing the fish-originated antifreeze protein (Type
III). In a concentration of 10 mg/ml or more, the sample containing
the fish-originated antifreeze protein (Type III) had a constant
freezing point of about -0.73.degree. C., whereas the sample
containing the Typhula ishikariensis-originated antifreeze protein
showed a certain freezing point depression in the concentration of
10 to 20 mg/ml, and the lowest value was -0.96.degree. C. The
result is shown FIG. 2 as the difference between the freezing point
and the melting point, or freezing point depression.
[0094] The above test result proves that the antifreeze protein of
the present invention provides a greater thermal hysteresis and a
higher antifreeze activity than those in the conventional
antifreeze protein.
Example 5
Cloning Typhula ishikariensis-Originated Antifreeze Protein
[0095] mRNA was extracted from 1 g of Typhula ishikariensis cells
cultured at 0.degree. C. or less for one month by using a RNA
adjustment kit (available from QIAGEN). Based on the obtained mRNA,
cDNA and cDNA library were prepared by using a cDNA preparation kit
(BD Bioscience Clontech).
[0096] Then, N-terminal and internal amino acid sequences of a
purified Typhula ishikariensis-originated antifreeze protein, and a
Typhula ishikariensis-originated antifreeze protein peptide
obtained through trypsin treatment, were determined by using a
protein sequencer (available from Applied Biosystem). Based on the
obtained sequences, a specific primer of the Typhula
ishikariensis-originated antifreeze protein was prepared. A part of
the DNA of the antifreeze protein was amplified through PCR
reaction with the cDNA template by using the designed specific
primer of the Typhula ishikariensis-originated antifreeze protein,
and a fragment of the amplified region was isolated. Further, the
polynucleotides encoding the antifreeze proteins of the present
invention were isolated from the cDNA library through a 3'/5'-RACE
method in their entire length. By checking with the Protein
Sequence Database, it was verified that all of seven, isolated
polynucleotides encoding antifreeze proteins were novel. The base
sequences of the seven polynucleotides are shown in SEQ ID NOS: 8,
10, 12, 14, 16, 18 and 20, respectively. The amino acid sequences
of proteins encoded by the polynucleotides are shown in SEQ ID NOS:
9, 11, 13, 15, 17, 19 and 21, respectively.
Example 6
Preparation of Typhula ishikariensis-Originated Antifreeze Protein
using Yeast Expression System
[0097] The polynucleotide encoding a Typhula
ishikariensis-originated antifreeze protein obtained through
cloning was inserted into the chromosome of methylotrophic yeast
(Pichia partoris) by using a methylotrophic yeast expression system
preparation kit (available from INVITROGEN). When the obtained
methylotrophic yeast transformant was cultured in BMMY medium at
25.degree. C. for five days, the Typhula ishikariensis-originated
antifreeze protein was excreted in the medium. A sample was taken
from the medium to check the ice crystal configuration therein. As
a result, a specific star-shaped ice crystal was observed in the
antifreeze protein. This means that the Typhula
ishikariensis-originated antifreeze protein prepared through the
gene recombination method exhibits a sufficient antifreeze
activity.
[0098] As mentioned above, the antifreeze protein originated from
basidiomycetes has a higher antifreeze activity, such as a thermal
hysteresis activity or an ice-recrystallization inhibition
activity, as compared to the conventional antifreeze proteins. In
addition, by taking advantage of biological properties of fungi,
the antifreeze protein of the present invention can be readily
cultured and prepared in large quantities at a low cost. It is
believed that the antifreeze protein of the present invention has a
high level of safety because no toxicity to human in all of the
tested basidiomycetes has been reported. Thus, the present
invention provides a significantly useful and valuable technology
for facilitating the utilization of antifreeze proteins as a
quality improving agent for frozen foods such as ice cream, a
cryopreservative for cells, or an additive for eliminating clogging
of pipelines due to freeze in cryogenic supply systems or cryogenic
storage systems.
Sequence CWU 1
1
21 1 20 PRT Typhula ishikariensis 1 Ala Gly Pro Ser Ala Val Ala Gly
Leu Thr Ala Gly Asn Tyr Ala Ile 1 5 10 15 Leu Ala Ser Thr 20 2 20
PRT Typhula ishikariensis 2 Ala Gly Pro Ser Ala Val Pro Leu Gly Thr
Ala Gly Asn Tyr Val Ile 1 5 10 15 Leu Ala Ser Thr 20 3 20 PRT
Typhula ishikariensis 3 Ala Gly Pro Thr Ala Val Pro Leu Gly Thr Ala
Gly Asn Tyr Ala Ile 1 5 10 15 Leu Ala Ser Thr 20 4 20 PRT Typhula
ishikariensis 4 Ala Gly Pro Ser Ala Val Pro Leu Gly Thr Ala Gly Asn
Tyr Ala Ile 1 5 10 15 Leu Ala Ser Thr 20 5 20 PRT Coprinus
psychromorbidus 5 Ala Gly Pro Thr Ala Val Asn Leu Gly Thr Ala Lys
Asn Tyr Ala Ile 1 5 10 15 Leu Thr Lys Ala 20 6 20 PRT Coprinus
psychromorbidus 6 Ala Gly Pro Thr Ala Val Asn Leu Gly Thr Ala Lys
Thr Tyr Ala Ile 1 5 10 15 Leu Thr Lys Ala 20 7 20 PRT Coprinus
psychromorbidus 7 Ala Gly Pro Thr Ala Val Asn Leu Gly Thr Ala Lys
Asn Tyr Ala Ile 1 5 10 15 Leu Thr Lys Thr 20 8 732 DNA Typhula
ishikariensis 8 atgttctcct caacctacct cctcgcaatc atcgccttgg
ctgtctcaag cgtttttgct 60 gctggtccca ccgctgtccc ccttggaacc
gccggcaact acgccattct cgcgtcggct 120 ggcgtttcga ctgtccccca
gtctgtcatc actggtgccg tcggactttc ccccgctgct 180 gcgactttcc
tcaccggatt tagtctcacg atgtctagca ccggcacctt ttccacgtca 240
actcaagtca ctggccagct tactgctgct gactatggta cgcctacccc tagtattttg
300 accactgcga tcggcgacat gggaactgcc tatgtcaacg cagctactcg
atcgggaccc 360 aactttctcg agatttacac tggggcactt ggcgggaaga
ttctccctcc tggtctatac 420 aaatggactt ctcccgtcgg tgcctccgct
gacttcacca ttattggtac atccaccgac 480 acctggatct tccaaattgc
tgggactctt ggactcgccg ctggaaagaa aatcatcctt 540 gctggtggag
ctcaggctaa gaacatcgtc tgggttgttg ctggcgctgt ctccatcgaa 600
gctggagcca agtttgaggg tgttatcctc gcaaaaactg ccgttaccct caagaccgga
660 tcctccctca acggaaggat tttgtcgcag actgccgttg ccttgcaaaa
ggctaccgtc 720 gtgcaaaagt ag 732 9 243 PRT Typhula ishikariensis 9
Met Phe Ser Ser Thr Tyr Leu Leu Ala Ile Ile Ala Leu Ala Val Ser 1 5
10 15 Ser Val Phe Ala Ala Gly Pro Thr Ala Val Pro Leu Gly Thr Ala
Gly 20 25 30 Asn Tyr Ala Ile Leu Ala Ser Ala Gly Val Ser Thr Val
Pro Gln Ser 35 40 45 Val Ile Thr Gly Ala Val Gly Leu Ser Pro Ala
Ala Ala Thr Phe Leu 50 55 60 Thr Gly Phe Ser Leu Thr Met Ser Ser
Thr Gly Thr Phe Ser Thr Ser 65 70 75 80 Thr Gln Val Thr Gly Gln Leu
Thr Ala Ala Asp Tyr Gly Thr Pro Thr 85 90 95 Pro Ser Ile Leu Thr
Thr Ala Ile Gly Asp Met Gly Thr Ala Tyr Val 100 105 110 Asn Ala Ala
Thr Arg Ser Gly Pro Asn Phe Leu Glu Ile Tyr Thr Gly 115 120 125 Ala
Leu Gly Gly Lys Ile Leu Pro Pro Gly Leu Tyr Lys Trp Thr Ser 130 135
140 Pro Val Gly Ala Ser Ala Asp Phe Thr Ile Ile Gly Thr Ser Thr Asp
145 150 155 160 Thr Trp Ile Phe Gln Ile Ala Gly Thr Leu Gly Leu Ala
Ala Gly Lys 165 170 175 Lys Ile Ile Leu Ala Gly Gly Ala Gln Ala Lys
Asn Ile Val Trp Val 180 185 190 Val Ala Gly Ala Val Ser Ile Glu Ala
Gly Ala Lys Phe Glu Gly Val 195 200 205 Ile Leu Ala Lys Thr Ala Val
Thr Leu Lys Thr Gly Ser Ser Leu Asn 210 215 220 Gly Arg Ile Leu Ser
Gln Thr Ala Val Ala Leu Gln Lys Ala Thr Val 225 230 235 240 Val Gln
Lys 10 732 DNA Typhula ishikariensis 10 atgttctccg catcctccct
tctcgctgtt attgcgttgg ctatctccag cgtctctgcc 60 gctggtccct
ctgctgtccc actcggaact gcgggaaact atgttattct cgcgtcgact 120
ggcgtttcga ctgtccccca gtctgtcatc actggcgccg tcggagtctc tcccggtact
180 gccgcttccc ttaccggctt cagccttata ctatctggca ccggcacctt
ctccacgtcg 240 tctcaggtca ctggccagct tactggtgcc gactacggga
cgcctactcc tagtattttg 300 accactgcga ttggcgacat gggaactgcc
tatattaacg cagctactcg atcgggaccc 360 gacttcctcg agatttacac
tggggcgctt ggcgggacga ctctccttcc tggtctatac 420 aagtggacct
cttccgttgg tgcctccgcc gactttacca ttagtggcac atccaccgac 480
acatggattt tccagattga cggcactctt gatgttgcaa ctgggaagca gatcactctc
540 gttggcggag ctcaggctaa gaacatcatc tgggtcgtag ctggagctgt
taacattgag 600 gttggggcaa agttcgaagg gaccatcctc gcaaaaactg
ccgtcacctt caagaccggt 660 tcatccctca acggaaggat tctggcgcag
acttctgtcg ctctgcagtc cgctaccatt 720 gtggaaaagt ag 732 11 243 PRT
Typhula ishikariensis 11 Met Phe Ser Ala Ser Ser Leu Leu Ala Val
Ile Ala Leu Ala Ile Ser 1 5 10 15 Ser Val Ser Ala Ala Gly Pro Ser
Ala Val Pro Leu Gly Thr Ala Gly 20 25 30 Asn Tyr Val Ile Leu Ala
Ser Thr Gly Val Ser Thr Val Pro Gln Ser 35 40 45 Val Ile Thr Gly
Ala Val Gly Val Ser Pro Gly Thr Ala Ala Ser Leu 50 55 60 Thr Gly
Phe Ser Leu Ile Leu Ser Gly Thr Gly Thr Phe Ser Thr Ser 65 70 75 80
Ser Gln Val Thr Gly Gln Leu Thr Gly Ala Asp Tyr Gly Thr Pro Thr 85
90 95 Pro Ser Ile Leu Thr Thr Ala Ile Gly Asp Met Gly Thr Ala Tyr
Ile 100 105 110 Asn Ala Ala Thr Arg Ser Gly Pro Asp Phe Leu Glu Ile
Tyr Thr Gly 115 120 125 Ala Leu Gly Gly Thr Thr Leu Leu Pro Gly Leu
Tyr Lys Trp Thr Ser 130 135 140 Ser Val Gly Ala Ser Ala Asp Phe Thr
Ile Ser Gly Thr Ser Thr Asp 145 150 155 160 Thr Trp Ile Phe Gln Ile
Asp Gly Thr Leu Asp Val Ala Thr Gly Lys 165 170 175 Gln Ile Thr Leu
Val Gly Gly Ala Gln Ala Lys Asn Ile Ile Trp Val 180 185 190 Val Ala
Gly Ala Val Asn Ile Glu Val Gly Ala Lys Phe Glu Gly Thr 195 200 205
Ile Leu Ala Lys Thr Ala Val Thr Phe Lys Thr Gly Ser Ser Leu Asn 210
215 220 Gly Arg Ile Leu Ala Gln Thr Ser Val Ala Leu Gln Ser Ala Thr
Ile 225 230 235 240 Val Glu Lys 12 732 DNA Typhula ishikariensis 12
atgttctccg catcctccct tctcgctgtt attgcgttgg ctgtctccag cgtctctgcc
60 gctggtccct ctgctgtccc actcggaact gcgggaaact atgttattct
cgcgtcgact 120 ggcgtttcga ctgtccccca gtctgtcatc actggcgccg
tcggagtctc tcccggtact 180 gccgcttccc ttaccggctt cagccttata
ctatctggca ccggcacctt ctccacgtcg 240 tctcaggtca ctggccagct
tactggtgcc gactacggga cgcctactcc tagtattttg 300 accactgcga
ttggcgacat gggaactgcc tatattaacg cagctactcg atcgggaccc 360
gacttcctcg agatttacac tggtgcgctt ggcgggacga ctctccttcc tggtctatac
420 aagtggacct cttccgttgg tgcctccgcc gactttacca ttagtggcac
atccaccgac 480 acatggattt tccagattga cggcactctt gatgttgcaa
ctgggaagca gatcactctc 540 gttggcggag ctcaggctaa gaacatcatc
tgggttgtag ctggagctgt taacattgag 600 gttggggcaa agttcgaagg
gaccatcctc gcaaaaactg ccgtcacctt caagaccggt 660 tcatccctca
acggaaggat tctggcgcag actgctgtcg ctctgcagtc cgctaccatt 720
gtggaaaagt ag 732 13 243 PRT Typhula ishikariensis 13 Met Phe Ser
Ala Ser Ser Leu Leu Ala Val Ile Ala Leu Ala Val Ser 1 5 10 15 Ser
Val Ser Ala Ala Gly Pro Ser Ala Val Pro Leu Gly Thr Ala Gly 20 25
30 Asn Tyr Val Ile Leu Ala Ser Thr Gly Val Ser Thr Val Pro Gln Ser
35 40 45 Val Ile Thr Gly Ala Val Gly Val Ser Pro Gly Thr Ala Ala
Ser Leu 50 55 60 Thr Gly Phe Ser Leu Ile Leu Ser Gly Thr Gly Thr
Phe Ser Thr Ser 65 70 75 80 Ser Gln Val Thr Gly Gln Leu Thr Gly Ala
Asp Tyr Gly Thr Pro Thr 85 90 95 Pro Ser Ile Leu Thr Thr Ala Ile
Gly Asp Met Gly Thr Ala Tyr Ile 100 105 110 Asn Ala Ala Thr Arg Ser
Gly Pro Asp Phe Leu Glu Ile Tyr Thr Gly 115 120 125 Ala Leu Gly Gly
Thr Thr Leu Leu Pro Gly Leu Tyr Lys Trp Thr Ser 130 135 140 Ser Val
Gly Ala Ser Ala Asp Phe Thr Ile Ser Gly Thr Ser Thr Asp 145 150 155
160 Thr Trp Ile Phe Gln Ile Asp Gly Thr Leu Asp Val Ala Thr Gly Lys
165 170 175 Gln Ile Thr Leu Val Gly Gly Ala Gln Ala Lys Asn Ile Ile
Trp Val 180 185 190 Val Ala Gly Ala Val Asn Ile Glu Val Gly Ala Lys
Phe Glu Gly Thr 195 200 205 Ile Leu Ala Lys Thr Ala Val Thr Phe Lys
Thr Gly Ser Ser Leu Asn 210 215 220 Gly Arg Ile Leu Ala Gln Thr Ala
Val Ala Leu Gln Ser Ala Thr Ile 225 230 235 240 Val Glu Lys 14 732
DNA Typhula ishikariensis 14 atgttctccg catcctccct tctcgctgtt
attgcgttgg ctgtctccag cgtctctgcc 60 gctggtccct ctgctgtccc
actcggaact gcgggaaact atgttattct cgcgtcgact 120 ggcgtttcga
ctgtccccca gtctgtcatc actggcgccg tcggagtctc tcccggtact 180
gccgcttccc ttaccggctt cagccttata ctatctggca ccggcacctt ctccacgtcg
240 tctcaggtca ctggccagct tactggtgcc gactacggga cgcctactcc
tagtattttg 300 accactgcga ttggcgacat gggaactgcc tatattaacg
cagctactcg atcgggaccc 360 gacttcctcg agatttacac tggtgcgctt
ggcgggacga ctctccttcc tggtctatac 420 aagtggacct cttccgttgg
tgcctccgcc gactttacca ttagtggcac atccaccgac 480 acatggattt
tccagattga cggcactctt gatgttgcaa ctgggaagca gatcactctc 540
gttggcggag ctcaggctaa gaacatcatc tgggttgtag ctggagctgt taacattgag
600 gttggggcaa agttcgaagg gaccatcctc gcaaaaactg ccgtcacctt
caagaccggt 660 tcatccctca acggaaggat tctggcgcag actgctgtcg
ctctgcagtc cgcgtccatt 720 gtggaaaagt ag 732 15 243 PRT Typhula
ishikariensis 15 Met Phe Ser Ala Ser Ser Leu Leu Ala Val Ile Ala
Leu Ala Val Ser 1 5 10 15 Ser Val Ser Ala Ala Gly Pro Ser Ala Val
Pro Leu Gly Thr Ala Gly 20 25 30 Asn Tyr Val Ile Leu Ala Ser Thr
Gly Val Ser Thr Val Pro Gln Ser 35 40 45 Val Ile Thr Gly Ala Val
Gly Val Ser Pro Gly Thr Ala Ala Ser Leu 50 55 60 Thr Gly Phe Ser
Leu Ile Leu Ser Gly Thr Gly Thr Phe Ser Thr Ser 65 70 75 80 Ser Gln
Val Thr Gly Gln Leu Thr Gly Ala Asp Tyr Gly Thr Pro Thr 85 90 95
Pro Ser Ile Leu Thr Thr Ala Ile Gly Asp Met Gly Thr Ala Tyr Ile 100
105 110 Asn Ala Ala Thr Arg Ser Gly Pro Asp Phe Leu Glu Ile Tyr Thr
Gly 115 120 125 Ala Leu Gly Gly Thr Thr Leu Leu Pro Gly Leu Tyr Lys
Trp Thr Ser 130 135 140 Ser Val Gly Ala Ser Ala Asp Phe Thr Ile Ser
Gly Thr Ser Thr Asp 145 150 155 160 Thr Trp Ile Phe Gln Ile Asp Gly
Thr Leu Asp Val Ala Thr Gly Lys 165 170 175 Gln Ile Thr Leu Val Gly
Gly Ala Gln Ala Lys Asn Ile Ile Trp Val 180 185 190 Val Ala Gly Ala
Val Asn Ile Glu Val Gly Ala Lys Phe Glu Gly Thr 195 200 205 Ile Leu
Ala Lys Thr Ala Val Thr Phe Lys Thr Gly Ser Ser Leu Asn 210 215 220
Gly Arg Ile Leu Ala Gln Thr Ala Val Ala Leu Gln Ser Ala Ser Ile 225
230 235 240 Val Glu Lys 16 732 DNA Typhula ishikariensis 16
atgttctcct caacctacct cctcgcaatc atcgccttgg ctatctcaag cgtttctgct
60 gctggaccca ccgctgtccc ccttggaacc gccggcaact acgccatcct
cgcgtcgacc 120 gctgtttcca ccgtccccca gtctgccatt actggtgccg
tcggaatttc ccccgctgct 180 gggactttcc ttaccggatt tagtctcacg
atgtctggca ccggcacctt ttccacgtca 240 actcaagtca ccggccagct
tactgctgct gactacggga cgcctacccc tagtatttta 300 accactgcga
ttggcgacat gggaactgcc tataccaacg gagctactcg atcgggaccc 360
gacttcctcg agatttacac tggggcgctt ggcgggacga ctctccttcc tggtctatac
420 aagtggacct cttccgttgg tgcctccgcc gactttacca ttagtggcac
atccaccgac 480 acatggattt tccaaattga cggcactctt ggactcgccg
ccggaaagaa aatcaccctt 540 gctggcggag ctcaggctaa gaacatcatc
tgggttgtag ctggagctgt tagcattgag 600 gctggagccc agttcgaggg
tgttatcctc gcaaaaactg ccgttactct caagaccgga 660 tcctccctca
acggaaggat tttggcgcag acttctgttg ctctgcagtc cgctaccgtc 720
gtgcaaaagt ag 732 17 243 PRT Typhula ishikariensis 17 Met Phe Ser
Ser Thr Tyr Leu Leu Ala Ile Ile Ala Leu Ala Ile Ser 1 5 10 15 Ser
Val Ser Ala Ala Gly Pro Thr Ala Val Pro Leu Gly Thr Ala Gly 20 25
30 Asn Tyr Ala Ile Leu Ala Ser Thr Ala Val Ser Thr Val Pro Gln Ser
35 40 45 Ala Ile Thr Gly Ala Val Gly Ile Ser Pro Ala Ala Gly Thr
Phe Leu 50 55 60 Thr Gly Phe Ser Leu Thr Met Ser Gly Thr Gly Thr
Phe Ser Thr Ser 65 70 75 80 Thr Gln Val Thr Gly Gln Leu Thr Ala Ala
Asp Tyr Gly Thr Pro Thr 85 90 95 Pro Ser Ile Leu Thr Thr Ala Ile
Gly Asp Met Gly Thr Ala Tyr Thr 100 105 110 Asn Gly Ala Thr Arg Ser
Gly Pro Asp Phe Leu Glu Ile Tyr Thr Gly 115 120 125 Ala Leu Gly Gly
Thr Thr Leu Leu Pro Gly Leu Tyr Lys Trp Thr Ser 130 135 140 Ser Val
Gly Ala Ser Ala Asp Phe Thr Ile Ser Gly Thr Ser Thr Asp 145 150 155
160 Thr Trp Ile Phe Gln Ile Asp Gly Thr Leu Gly Leu Ala Ala Gly Lys
165 170 175 Lys Ile Thr Leu Ala Gly Gly Ala Gln Ala Lys Asn Ile Ile
Trp Val 180 185 190 Val Ala Gly Ala Val Ser Ile Glu Ala Gly Ala Gln
Phe Glu Gly Val 195 200 205 Ile Leu Ala Lys Thr Ala Val Thr Leu Lys
Thr Gly Ser Ser Leu Asn 210 215 220 Gly Arg Ile Leu Ala Gln Thr Ser
Val Ala Leu Gln Ser Ala Thr Val 225 230 235 240 Val Gln Lys 18 732
DNA Typhula ishikariensis 18 atgttctccg catcctccct tctcgctgtt
attgcgttga ctatctccag cgtctctgcc 60 gctggtccct ctgctgtccc
actcggaact gcgggaaact atgttattct cgcgtcgact 120 ggcgtttcga
ctgtccccca gtctgtcatc actggcgccg tcggagtctc tcccggtact 180
gccgcttccc ttaccggctt cagccttata ctatctggca ccggcacctt ctccacgtcg
240 tctcaggtca ctggccagct tactggtgcc gactacggga cgcctactcc
tagtattttg 300 accactgcga ttggcgacat gggaactgcc tatattaacg
cagctactcg atcgggaccc 360 gacttcctcg agatttacac tggggcgctt
ggcgggacga ctctccttcc tggtctatac 420 aagtggacct cttccgttgg
tgcctccgcc gactttacca ttagtggcac atccaccgac 480 acatggattt
tccagattga cggcactctt gatgttgcaa ctgggaagca gatcactctc 540
gttggcggag ctcaggctaa gaacgtcatc tgggttgtag ctggagctgt taacattgag
600 gttggggcaa agttcgaagg gaccatcctc gcaaaaactg ccgtcacctt
caagaccggt 660 tcatccctca acggaaggat tctggcgcag actgctgtcg
ctctgcagtc cgctaccatt 720 gtggaaaagt ag 732 19 243 PRT Typhula
ishikariensis 19 Met Phe Ser Ala Ser Ser Leu Leu Ala Val Ile Ala
Leu Thr Ile Ser 1 5 10 15 Ser Val Ser Ala Ala Gly Pro Ser Ala Val
Pro Leu Gly Thr Ala Gly 20 25 30 Asn Tyr Val Ile Leu Ala Ser Thr
Gly Val Ser Thr Val Pro Gln Ser 35 40 45 Val Ile Thr Gly Ala Val
Gly Val Ser Pro Gly Thr Ala Ala Ser Leu 50 55 60 Thr Gly Phe Ser
Leu Ile Leu Ser Gly Thr Gly Thr Phe Ser Thr Ser 65 70 75 80 Ser Gln
Val Thr Gly Gln Leu Thr Gly Ala Asp Tyr Gly Thr Pro Thr 85 90 95
Pro Ser Ile Leu Thr Thr Ala Ile Gly Asp Met Gly Thr Ala Tyr Ile 100
105 110 Asn Ala Ala Thr Arg Ser Gly Pro Asp Phe Leu Glu Ile Tyr Thr
Gly 115 120 125 Ala Leu Gly Gly Thr Thr Leu Leu Pro Gly Leu Tyr Lys
Trp Thr Ser 130 135 140 Ser Val Gly Ala Ser Ala Asp Phe Thr Ile Ser
Gly Thr Ser Thr Asp 145 150 155 160 Thr Trp Ile Phe Gln Ile Asp Gly
Thr Leu Asp Val Ala Thr Gly Lys 165 170 175 Gln Ile Thr Leu Val Gly
Gly Ala Gln Ala Lys Asn Val Ile Trp Val 180 185 190 Val Ala Gly Ala
Val Asn Ile Glu Val Gly Ala Lys Phe Glu Gly Thr 195 200 205 Ile Leu
Ala Lys Thr Ala Val Thr Phe Lys Thr Gly Ser Ser Leu Asn 210 215 220
Gly Arg Ile Leu Ala Gln Thr Ala Val Ala Leu Gln Ser Ala Thr Ile 225
230 235 240 Val Glu
Lys 20 732 DNA Typhula ishikariensis 20 atgttctccg catcctccct
tctcgctgtt attgcgttgg ctatctccag cgtctctgcc 60 gctggtccct
ctgctgtccc actcggaact gcgggaaact atgttattct cgcgtcgact 120
ggcgtttcga ctgtccccca gtctgtcatc actggcgccg tcggagtctc tcccggtact
180 gccgcttccc ttaccggctt cagccttata ctatctggca ccggcacctt
ctccacgtcg 240 tctcaggtca ctggccagct tactggtgct gactacggga
cgcctactcc tagtattttg 300 accactgcga ttggcgacat gggaactgcc
tatattaacg cagctactcg atcgggaccc 360 gacttcctcg agatttacac
tggggcgctt ggcgggacga ctctccttcc tggtctatac 420 aagtggacct
cttccgttgg tgcctccgcc gactttacca ttagtggcac atccaccgac 480
acatggattt tccaaattga cggcactctt ggactcgccg ccggaaagaa aatcactctc
540 gttggcggag ctcaggctaa gaacgtcatc tgggttgtag ctggagctgt
taacattgag 600 gttggggcaa agttcgaagg gaccatcctc gcaaaaactg
ccgtcacctt caagaccggt 660 tcatccctca acggaaggat tctggcgcag
actgctgtcg ctctgcagtc cgctaccatt 720 gtggaaaagt ag 732 21 243 PRT
Typhula ishikariensis 21 Met Phe Ser Ala Ser Ser Leu Leu Ala Val
Ile Ala Leu Ala Ile Ser 1 5 10 15 Ser Val Ser Ala Ala Gly Pro Ser
Ala Val Pro Leu Gly Thr Ala Gly 20 25 30 Asn Tyr Val Ile Leu Ala
Ser Thr Gly Val Ser Thr Val Pro Gln Ser 35 40 45 Val Ile Thr Gly
Ala Val Gly Val Ser Pro Gly Thr Ala Ala Ser Leu 50 55 60 Thr Gly
Phe Ser Leu Ile Leu Ser Gly Thr Gly Thr Phe Ser Thr Ser 65 70 75 80
Ser Gln Val Thr Gly Gln Leu Thr Gly Ala Asp Tyr Gly Thr Pro Thr 85
90 95 Pro Ser Ile Leu Thr Thr Ala Ile Gly Asp Met Gly Thr Ala Tyr
Ile 100 105 110 Asn Ala Ala Thr Arg Ser Gly Pro Asp Phe Leu Glu Ile
Tyr Thr Gly 115 120 125 Ala Leu Gly Gly Thr Thr Leu Leu Pro Gly Leu
Tyr Lys Trp Thr Ser 130 135 140 Ser Val Gly Ala Ser Ala Asp Phe Thr
Ile Ser Gly Thr Ser Thr Asp 145 150 155 160 Thr Trp Ile Phe Gln Ile
Asp Gly Thr Leu Gly Leu Ala Ala Gly Lys 165 170 175 Lys Ile Thr Leu
Val Gly Gly Ala Gln Ala Lys Asn Val Ile Trp Val 180 185 190 Val Ala
Gly Ala Val Asn Ile Glu Val Gly Ala Lys Phe Glu Gly Thr 195 200 205
Ile Leu Ala Lys Thr Ala Val Thr Phe Lys Thr Gly Ser Ser Leu Asn 210
215 220 Gly Arg Ile Leu Ala Gln Thr Ala Val Ala Leu Gln Ser Ala Thr
Ile 225 230 235 240 Val Glu Lys
* * * * *