U.S. patent application number 13/441118 was filed with the patent office on 2012-10-18 for spider silk proteins and methods for producing spider silk proteins.
Invention is credited to Wilhelm Engstrom, Stefan Grip, My Hedhammar, Goran Hjalm, Jan JOHANSSON, Anna Rising, Margareta Stark.
Application Number | 20120264914 13/441118 |
Document ID | / |
Family ID | 37836847 |
Filed Date | 2012-10-18 |
United States Patent
Application |
20120264914 |
Kind Code |
A1 |
JOHANSSON; Jan ; et
al. |
October 18, 2012 |
SPIDER SILK PROTEINS AND METHODS FOR PRODUCING SPIDER SILK
PROTEINS
Abstract
The invention provides an isolated major ampullate spidroin
protein, which consists of from 150 to 420 amino acid residues and
is defined by the formula REP-CT. REP is a repetitive, N-terminally
derived protein fragment having from 80 to 300 amino acid residues.
CT is a C-terminally derived protein fragment having from 70 to 120
amino acid residues. The invention further provides an isolated
fusion protein consisting of a first protein fragment, which is a
major ampullate spidroin protein, and a second protein fragment
comprising a fusion partner and a cleavage agent recognition site.
The first protein fragment is coupled via said cleavage agent
recognition site to the fusion partner. The invention also provides
a method of producing a major ampullate spidroin protein and
polymers thereof.
Inventors: |
JOHANSSON; Jan; (Stockholm,
SE) ; Hjalm; Goran; (Uppsala, SE) ; Stark;
Margareta; (Hagersten, SE) ; Rising; Anna;
(Uppsala, SE) ; Grip; Stefan; (Uppsala, SE)
; Engstrom; Wilhelm; (Saltsjo-Boo, SE) ;
Hedhammar; My; (Stockholm, SE) |
Family ID: |
37836847 |
Appl. No.: |
13/441118 |
Filed: |
April 6, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12087289 |
Jun 30, 2008 |
8173772 |
|
|
PCT/SE2006/001505 |
Dec 28, 2006 |
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13441118 |
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Current U.S.
Class: |
530/353 ;
435/68.1; 435/69.7; 536/23.1 |
Current CPC
Class: |
A61K 35/12 20130101;
A61K 47/42 20130101; C07K 14/43518 20130101; D01F 4/00
20130101 |
Class at
Publication: |
530/353 ;
536/23.1; 435/69.7; 435/68.1 |
International
Class: |
C07K 14/435 20060101
C07K014/435; C12P 21/02 20060101 C12P021/02; C12P 21/06 20060101
C12P021/06; C12N 15/12 20060101 C12N015/12 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 30, 2005 |
SE |
0502932-7 |
Claims
1. An isolated major ampullate spidroin protein, consisting of from
150 to 420 amino acid residues and is defined by the formula
REP-CT, wherein REP is a protein fragment having from 80 to 300
amino acid residues, wherein said fragment is selected from the
group of L(AG).sub.nL (SEQ ID NO: 17), L(AG).sub.nAL (SEQ ID NO:
18), L(GA).sub.nL (SEQ ID NO: 19), L(GA).sub.nGL (SEQ ID NO: 120),
wherein n is an integer from 4 to 8; each individual A segment is
an amino acid sequence of from 8 to 18 amino acid residues, wherein
from 0 to 3 of the amino acid residues are not Ala, and the
remaining amino acid residues are Ala; each individual G segment is
an amino acid sequence of from 12 to 30 amino acid residues,
wherein at least 40% of the amino acid residues are Gly; and each
individual L segment is a linker amino acid sequence of from 0 to
20 amino acid residues; and CT is a protein fragment having from 70
to 120 amino acid residues, which fragment is a C-terminal fragment
derived from a major ampullate spidroin protein, wherein said CT
fragment is, or has at least 80% identity to, an amino acid
sequence selected from the group consisting of SEQ ID NO: 4, amino
acid residues 172-269 of SEQ ID NO: 9, amino acid residues 181-276
of SEQ ID NO: 13, and amino acid residues 172-269 of SEQ ID NO:
16.
2. The isolated protein according to claim 1, wherein each
individual A segment is, or has at least 80% identity to, an amino
acid sequence selected from the group of amino acid residues 7-19,
43-56, 71-83, 107-120, 135-147, 171-183, 198-211, 235-248, 266-279,
294-306, 330-342, 357-370, 394-406, 421-434, 458-470, 489-502,
517-529, 553-566, 581-594, 618-630, 648-661, 676-688, 712-725,
740-752, 776-789, 804-816, 840-853, 868-880, 904-917, 932-945,
969-981, 999-1013, 1028-1042 and 1060-1073 of SEQ ID NO: 3; amino
acid residues 31-42, 61-75, 90-104, 122-135 and 153-171 of SEQ ID
NO: 9; amino acid residues 12-25, 46-60, 75-88, 112-119, 150-158
and 173-180 of SEQ ID NO: 13; amino acid residues 31-42 of SEQ ID
NO: 14; and amino acid residues 122-135 of SEQ ID NO: 15; and each
individual G segment is identical to, or has at least 80% identity
to, an amino acid sequence selected from the group of amino acid
residues 20-42, 57-70, 84-106, 121-134, 148-170, 184-197, 212-234,
249-265, 280-293, 307-329, 343-356, 371-393, 407-420, 435-457,
471-488, 503-516, 530-552, 567-580, 595-617, 631-647, 662-675,
689-711, 726-739, 753-775, 790-803, 817-839, 854-867, 881-903,
918-931, 946-968, 982-998, 1014-1027, 1043-1059 and 1074-1092 of
SEQ ID NO: 3; SEQ ID NOS: 5-7; amino acid residues 11-30, 43-60,
76-89, 105-121 and 136-152 of SEQ ID NO: 9; and amino acid residues
1-11, 26-45, 61-74, 89-111, 120-149 and 159-172 of SEQ ID NO:
13.
3. An isolated fusion protein, comprising: the protein according to
claim 1; and a protein fragment comprising a fusion partner and a
cleavage agent recognition site, wherein the protein is coupled via
said cleavage agent recognition site to said fusion partner.
4. A polymer of a major ampullate spidroin protein according to
claim 1.
5. A composition comprising the isolated protein according to claim
1, wherein the content of lipopolysaccharide (LPS) and other
pyrogens is 1 EU/mg of isolated protein or lower.
6. An isolated nucleic acid molecule comprising a nucleic acid
sequence selected from the group consisting of SEQ ID NO: 1;
nucleic acid sequences encoding SEQ ID NOS: 2-16, and their
complementary nucleic acid sequences; and nucleic acid sequences
which encodes a fusion protein according to claim 3, and their
complementary nucleic acid sequences.
7. A method of producing a soluble fusion protein as defined in
claim 3, comprising the steps of: (i) expressing a nucleic acid
molecule which encodes the fusion protein according to claim 32 in
a suitable host; and (ii) isolating the soluble fusion protein
obtained in step (i), optionally involving removal of
lipopolysaccharide (LPS) and other pyrogens.
8. A method of producing a polymer of a major ampullate spidroin
protein, comprising the steps of: (i) providing a solution of the
fusion protein according to claim 3 in a liquid medium, (ii) adding
to said liquid medium a suitable cleaving agent for achieving
cleavage of the fusion protein at the cleavage agent recognition
site, and thereby obtaining the major ampullate spidroin protein;
(iii) allowing the major ampullate spidroin protein obtained in
step (ii) to polymerize in the liquid medium; and optionally (iv)
isolating the polymer obtained in step (iii) from said liquid
medium, optionally involving removal of lipopolysaccharide (LPS)
and other pyrogens.
9. The method according to claim 8, wherein step (i) comprises: (a)
expressing a nucleic acid molecule which encodes said fusion
protein in a suitable host; (b) isolating the soluble fusion
protein obtained in step (a), optionally involving removal of
lipopolysaccharide (LPS) and other pyrogens; and (c) providing a
solution of said soluble fusion protein obtained in step (b) in a
liquid medium.
10. The method according to claim 8, wherein said step (iii) of
allowing the major ampullate spidroin protein obtained in step (ii)
to polymerize in the liquid medium, further comprises providing an
interface between said liquid medium and another phase selected
from the group consisting of a gas phase, a liquid phase and a
solid phase, wherein said polymerizing initiates at said interface
or in a region surrounding said interface.
11. The method according to claim 10, wherein said liquid medium is
an aqueous medium and said other phase is selected from the group
consisting of air and water-immiscible organic solvents.
12. The isolated protein according to claim 1, wherein said protein
has at least 80% identity to an amino acid sequence selected from
amino acid residues 10-269 of SEQ ID NO: 9 and amino acid residues
1-276 of SEQ ID NO: 13.
Description
[0001] This application is a Continuation of co-pending application
Ser. No. 12/087,289 filed Jun. 30, 2008, which is the National
phase of PCT International Application No. PCT/SE2006/001505 filed
on Dec. 28, 2006. This application also claims priority to Patent
Application No. 0502932-7 filed in Sweden on Dec. 30, 2005. All of
the above applications are hereby expressly incorporated by
reference into the present application.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to the field of recombinant
production of proteins. More specifically, the present invention is
concerned with recombinant production of spider silk proteins. The
present invention provides novel isolated major ampullate spidroin
proteins and major ampullate spidroin fusion proteins, as well as
methods and polynucleic acid molecules for producing such proteins.
There is also provided polymers of the major ampullate spidroin
proteins and methods for producing such polymers.
BACKGROUND OF THE INVENTION
[0003] Spider silks are nature's high-performance polymers,
obtaining extraordinary toughness due to a combination of strength
and elasticity. Up to seven specialized glands exist in spiders,
which produce a variety of silk fiber types with different
mechanical properties and functions. Dragline silk, produced by the
major ampullate gland, is the toughest fiber, and on a weight basis
it outperforms man-made materials, such as high tensile steel and
Kevlar. The properties of dragline silk are attractive in
development of new materials for medical or technical purposes.
[0004] Dragline silk consists of two main polypeptides, mostly
referred to as major ampullate spidroin (MaSp) 1 and 2, but to
ADF-3 and ADF-4 in Araneus diadematus. These proteins have apparent
molecular masses in the range of 200-720 kDa, depending on sample
age and conditions of analysis, but no full-length dragline spider
silk gene has yet been reported. The properties of dragline silk
polypeptides are discussed in Huemmerich, D. et al. Novel assembly
properties of recombinant spider dragline silk proteins. Curr.
Biol. 14, 2070-2074 (2004). The known dragline silk spidroins are
composed of highly iterated blocks of alternating alanine-rich
segments, forming crystalline .beta.-sheets in the fiber, and
glycine-rich segments which are more flexible and mainly lack
ordered structure. The C-terminal region is non-repetitive, highly
conserved between species, and adopts .alpha.-helical conformation.
The N-terminal region of dragline silk proteins has not been
characterized until very recently, revealing an N-terminal domain
that is highly conserved between different spidroins, and also
between different spider species (Rising, A. et al. N-terminal
nonrepetitive domain common to dragline, flagelliform, and
cylindriform spider silk proteins. Biomacromolecules 7, 3120-3124
(2006)).
[0005] The mechanical properties of dragline silk varies between
species; Euprosthenops sp dragline silk is stiffer, stronger
(requires more force to break) and less extendible than dragline
silk from e.g. Araneus diadematus or Nephila clavipes. Dragline
silk from Euprosthenops sp appears to have a greater proportion of
crystalline .beta.-sheet structure than dragline silk from Araneus
diadematus, most likely due to that the Euprosthenops sp MaSp has
the highest polyalanine content among all species analyzed so far
(Pouchkina-Stantcheva, N. N. & McQueen-Mason, S. J. Molecular
studies of a novel dragline silk from a nursery web spider,
Euprosthenops sp. (Pisauridae). Comp Biochem Physiol B Biochem Mol
Biol 138, 371-376 (2004)).
[0006] Attempts to produce artificial spider silks have employed
natural or synthetic gene fragments encoding dragline silk
proteins, since no full-length gene has yet been reported.
Recombinant dragline silk proteins have been expressed in various
systems including bacteria, yeast, mammalian cells, plants, insect
cells, transgenic silkworms and transgenic goats. See e.g. Lewis,
R. V. et al. Expression and purification of a spider silk protein:
a new strategy for producing repetitive proteins. Protein Expr.
Purif. 7, 400-406 (1996); Fahnestock, S. R. & Irwin, S. L.
Synthetic spider dragline silk proteins and their production in
Escherichia coli. Appl. Microbiol. Biotechnol. 47, 23-32 (1997);
Arcidiacono, S. et al. Purification and characterization of
recombinant spider silk expressed in Escherichia coli. Appl.
Microbiol. Biotechnol. 49, 31-38 (1998); Fahnestock, S. R. &
Bedzyk, L. A. Production of synthetic spider dragline silk protein
in Pichia pastoris. Appl. Microbiol. Biotechnol. 47, 33-39 (1997);
and Lazaris, A. et al. Spider silk fibers spun from soluble
recombinant silk produced in mammalian cells. Science 295, 472-476
(2002).
[0007] WO 2004/016651 (The University of York) discloses nucleic
acid sequences coding for internal, repetitive parts of MaSp1
proteins from Euprosthenops sp. No protein is expressed.
[0008] Huemmerich, D. et al. Primary structure elements of spider
dragline silks and their contribution to protein solubility.
Biochemistry 43, 13604-13612 (2004) discloses a synthetic gene,
"(AQ).sub.12NR3", coding for repetitive Ala-rich and Gly/Gln-rich
fragments and a non-repetitive fragment, all derived from ADF3 from
Araneus. The gene is expressed into a soluble protein (59.8 kD,
>528 aa), which aggregates but does not form polymers or fibers.
The alanine content of the protein is 10-15%.
[0009] WO 03/057727 discloses expression of soluble recombinant
silk polypeptides in mammalian cell lines and animals. One
expressed silk polypeptide (ADF-3; 60 kD, 652 aa) consists of a
repetitive unit and a non-repetitive hydrophilic domain. Another
expressed silk polypeptide (ADF-3 His; 63 kD, 677 aa) consists of a
repetitive unit, a non-repetitive hydrophilic domain, a c-myc
epitope and a six-Histidine tag. The repetitive unit has a low
content of Ala (10-20%). The obtained silk polypeptides exhibit
poor solubility in aqueous media and/or form precipitates. Since
the obtained silk polypeptides do not polymerize spontaneously,
spinning is required to obtain polymers or fibers.
[0010] Several factors complicate the expression of dragline silk
proteins. Due to the highly repetitive nature of the genes, and the
concomitant restricted amino acid composition of the proteins,
transcription and translation errors occur. Depletion of tRNA-pools
in microbial expression systems, with subsequent discontinuous
translation, leading to premature termination of protein synthesis
might be another reason. Other reasons discussed for truncation of
protein synthesis are secondary structure formation of the mRNA,
and recombination of the genes. Native MaSp genes larger than 2.5
kb have been shown to be instable in bacterial hosts. Additionally,
there are difficulties in maintaining the recombinant silk proteins
in soluble form, since both natural-derived dragline silk fragments
and designed block copolymers, especially MaSp1/ADF-4-derived
proteins, easily self-assemble into amorphous aggregates, causing
precipitation and loss of protein. See Huemmerich, D. et al.
Primary structure elements of spider dragline silks and their
contribution to protein solubility. Biochemistry 43, 13604-13612
(2004) and Lazaris, A. et al. Spider silk fibers spun from soluble
recombinant silk produced in mammalian cells. Science 295, 472-476
(2002).
SUMMARY OF THE INVENTION
[0011] It is an object of the present invention to provide a novel
spider silk protein, which can provide spider silk fibers.
[0012] It is another object of the present invention to provide a
water-soluble spider silk protein, which can readily be manipulated
to self-polymerize into fibers at wish. This allows for unique
applications, such as culturing of eukaryotic cells on the fibers.
Furthermore, this property allows for all the following steps to be
undertaken under physiological conditions, which decreases the risk
for toxicity and protein denaturation.
[0013] It is yet another object of the present invention to provide
fibers of a novel spider silk protein.
[0014] It is one object of the present invention to provide spider
silk proteins in large scale, which proteins can readily be
manipulated to self-polymerize into fibers at wish.
[0015] It is also an object of the invention to provide methods of
producing silk proteins and fibers of spider silk proteins.
[0016] For these and other objects that will be evident from the
following disclosure, the present invention provides according to
one aspect an isolated major ampullate spidroin protein, wherein
the protein consists of from 150 to 420 amino acid residues and is
defined by the formula REP-CT, wherein REP is a protein fragment
having from 80 to 300 amino acid residues, wherein said fragment is
selected from the group of L(AG).sub.nL (SEQ ID NO: 17),
L(AG).sub.nAL (SEQ ID NO: 18), L(GA).sub.nL (SEQ ID NO: 19),
L(GA).sub.nGL (SEQ ID NO: 20), wherein n is an integer from 4 to
8;
each individual A segment is an amino acid sequence of from 8 to 18
amino acid residues, wherein from 0 to 3 of the amino acid residues
are not Ala, and the remaining amino acid residues are Ala; each
individual G segment is an amino acid sequence of from 12 to 30
amino acid residues, wherein at least 40% of the amino acid
residues are Gly; and each individual L segment is a linker amino
acid sequence of from 0 to 20 amino acid residues; and CT is a
protein fragment having from 70 to 120 amino acid residues, which
fragment is a C-terminal fragment derived from a major ampullate
spidroin protein, or a derivative thereof.
[0017] The present invention is based on the identification of a
protein motif, which is sufficient to form silk-like fibers, and
the use of said motif for construction of recombinant MaSp
proteins, which are possible to produce in suitable hosts, such as
bacteria, preferably E. coli.
[0018] In certain embodiments according to the invention, each
individual A segment has at least 80% identity to an amino acid
sequence selected from the group of amino acid residues 7-19,
43-56, 71-83, 107-120, 135-147, 171-183, 198-211, 235-248, 266-279,
294-306, 330-342, 357-370, 394-406, 421-434, 458-470, 489-502,
517-529, 553-566, 581-594, 618-630, 648-661, 676-688, 712-725,
740-752, 776-789, 804-816, 840-853, 868-880, 904-917, 932-945,
969-981, 999-1013, 1028-1042 and 1060-1073 of SEQ ID NO: 3; amino
acid residues 31-42, 61-75, 90-104, 122-135 and 153-171 of SEQ ID
NO: 9; amino acid residues 12-25, 46-60, 75-88, 112-119, 150-158
and 173-180 of SEQ ID NO: 13; amino acid residues 31-42 of SEQ ID
NO: 14; and amino acid residues 122-135 of SEQ ID NO: 15. In
specific embodiments, each individual A segment is an amino acid
sequence selected from this group of amino acid sequences.
[0019] In some embodiments according to the invention, each
individual G segment has at least 80% identity to an amino acid
sequence selected from the group of amino acid residues 20-42,
57-70, 84-106, 121-134, 148-170, 184-197, 212-234, 249-265,
280-293, 307-329, 343-356, 371-393, 407-420, 435-457, 471-488,
503-516, 530-552, 567-580, 595-617, 631-647, 662-675, 689-711,
726-739, 753-775, 790-803, 817-839, 854-867, 881-903, 918-931,
946-968, 982-998, 1014-1027, 1043-1059 and 1074-1092 of SEQ ID NO:
3; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; amino acid residues
11-30, 43-60, 76-89, 105-121 and 136-152 of SEQ ID NO: 9; and amino
acid residues 1-11, 26-45, 61-74, 89-111, 120-149 and 159-172 of
SEQ ID NO: 13. In specific embodiments, each individual G segment
is identical to an amino acid sequence selected from this group of
amino acid sequences.
[0020] In certain embodiments according to the invention, said CT
fragment has at least 50% identity to SEQ ID NO: 8 or at least 80%
identity to an amino acid sequence selected from the group
consisting of SEQ ID NO: 4, amino acid residues 172-269 of SEQ ID
NO: 9, amino acid residues 181-276 of SEQ ID NO: 13 and amino acid
residues 172-269 of SEQ ID NO: 16 as well as any amino acid
sequence of FIG. 3, in particular the MaSp1 sequences of FIG. 3. In
specific embodiments, said CT fragment is an amino acid sequence
selected from this group of amino acid sequences.
[0021] In certain embodiments according to the invention, the
content of lipopolysaccharides (LPS) and other pyrogens in the
isolated major ampullate spidroin protein is 1 endotoxin unit
(EU)/mg protein or lower.
[0022] According to another aspect, the present invention provides
an isolated fusion protein consisting of a first protein fragment,
which is a major ampullate spidroin protein, and a second protein
fragment, wherein said second protein fragment comprises a fusion
partner and a cleavage agent recognition site, wherein said first
protein fragment is coupled via said cleavage agent recognition
site to said fusion partner.
[0023] The present invention provides an isolated fusion protein
selected from the group of X-REP-CT, and REP-CT-X, wherein REP and
CT are protein fragments according to the invention; and X is a
protein fragment comprising a fusion partner and a cleavage agent
recognition site; wherein the combined protein fragment REP-CT is
coupled via said cleavage agent recognition site to said fusion
partner.
[0024] In certain embodiments according to the invention, the
content of LPS and other pyrogens in the isolated fusion protein is
1 EU/mg protein or lower.
[0025] According to yet another aspect, the present invention
provides a method of producing a major ampullate spidroin protein
according to the invention, comprising the steps of: (i) providing
a solution of a fusion protein according to the invention in a
liquid medium, (ii) adding to said liquid medium a suitable
cleaving agent for achieving cleavage of the fusion protein at the
cleavage agent recognition site, and thereby obtaining the major
ampullate spidroin protein; and optionally (iii) isolating the
major ampullate spidroin protein obtained in step (ii) from said
liquid medium.
[0026] The present invention also provides a method of producing a
polymer of a major ampullate spidroin protein according to the
invention, comprising the steps of: (i) providing a solution of a
fusion protein according to the invention in a liquid medium, (ii)
adding to said liquid medium a suitable cleaving agent for
achieving cleavage of the fusion protein at the cleavage agent
recognition site, and thereby obtaining the major ampullate
spidroin protein; (iii) allowing the major ampullate spidroin
protein obtained in step (ii) to polymerize in the liquid medium;
and optionally (iv) isolating the polymer obtained in step (iii)
from said liquid medium. In a preferred method, said step (iii)
further comprises providing an interface between said liquid medium
and another phase selected from the group consisting of a gas
phase, a liquid phase and a solid phase, wherein said polymerizing
initiates at said interface or in a region surrounding said
interface. In a preferred method, said liquid medium is an aqueous
medium and said other phase is selected from the group consisting
of air and water-immiscible organic solvents.
[0027] According to another aspect, the present invention provides
an isolated polynucleic acid molecule comprising a nucleic acid
sequence which encodes a major ampullate spidroin protein according
to the invention, or its complementary nucleic acid sequence.
[0028] According to yet another aspect, the present invention
provides an isolated polynucleic acid molecule comprising a nucleic
acid sequence which encodes a fusion protein according to the
invention, or its complementary nucleic acid sequence.
[0029] Another aspect of the invention resides in a method of
producing a soluble fusion protein according to the invention,
comprising the steps of: (i) expressing a polynucleic acid molecule
encoding a soluble fusion protein according to the invention in a
suitable host; and (ii) isolating the soluble fusion protein
obtained in step (i). Optionally, said step (ii) of isolating the
soluble fusion protein involves removal of LPS and other
pyrogens.
[0030] The present invention also provides a method of producing a
major ampullate spidroin protein according to the invention,
comprising the steps of: (i) expressing a polynucleic acid molecule
encoding a soluble fusion protein according to the invention in a
suitable host; (ii) isolating the soluble fusion protein obtained
in step (i); (iii) providing a solution of said soluble fusion
protein obtained in step (ii) in a liquid medium, (iv) adding to
said liquid medium a suitable cleaving agent for achieving cleavage
of the fusion protein at the cleavage agent recognition site, and
thereby obtaining the major ampullate spidroin protein; and
optionally (v) isolating the major ampullate spidroin protein
obtained in step (iv) from said liquid medium. Further optionally,
said step (ii) of isolating the soluble fusion protein, and
optionally step (v) of isolating the major ampullate spidroin
protein, involve(s) removal of LPS and other pyrogens.
[0031] The present invention further provides a method of producing
a polymer of a major ampullate spidroin protein according to the
invention, comprising the steps of: (i) expressing a polynucleic
acid molecule encoding a soluble fusion protein according to the
invention in a suitable host; (ii) isolating the soluble fusion
protein obtained in step (i); (iii) providing a solution of said
soluble fusion protein obtained in step (ii) in a liquid medium,
(iv) adding to said liquid medium a suitable cleaving agent for
achieving cleavage of the fusion protein at the cleavage agent
recognition site, and thereby obtaining the major ampullate
spidroin protein; (v) allowing the major ampullate spidroin protein
obtained in step (iv) to polymerize in the liquid medium; and
optionally (vi) isolating the polymer obtained in step (v) from
said liquid medium. In a preferred method, said step (v) further
comprises providing an interface between said liquid medium and
another phase selected from the group consisting of a gas phase, a
liquid phase and a solid phase, wherein said polymerizing initiates
at said interface or in a region surrounding said interface. In a
preferred method, said liquid medium is an aqueous medium and said
other phase is selected from the group consisting of air and
water-immiscible organic solvents.
[0032] According to another aspect, the present invention provides
a polymer of a major ampullate spidroin protein according to the
invention. The present invention also provides a polymer of a major
ampullate spidroin protein obtainable by a method according to the
invention. In a preferred embodiment, said polymer is a fiber. In
other preferred embodiments, said polymer forms a structure
selected from the group consisting of a foam, a gel, a mesh or a
film.
[0033] According to yet another aspect, the present invention
provides a novel use of a protein fragment comprising a fusion
partner and a cleavage agent recognition site for the manufacture
of a fusion protein comprising said protein fragment coupled via
said cleavage agent recognition site to a spider silk protein
fragment. In preferred embodiments, said spider silk protein
fragment consists of from 150 to 420 amino acid residues.
[0034] According to a final aspect, the present invention provides
an isolated polynucleic acid molecule comprising a nucleic acid
sequence selected from the group consisting of SEQ ID NO: 1 and
nucleic acid sequences encoding SEQ ID NOS: 2-16, or its
complementary nucleic acid sequences. The present invention also
provides use of the isolated polynucleic acid molecule for the
manufacture of a non-natural gene encoding a spider silk
protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 is an alignment of the segments within the repetitive
part of Euprosthenops australis MaSp1 protein, i.e. SEQ ID NO:
3.
[0036] FIG. 2A illustrates a schematic, predicted structural
organization of the repetitive part of Euprosthenops australis
MaSp1 protein (SEQ ID NO: 3). The various peptide segments shown
correspond to SEQ ID NOS: 21-23 and 5-6, respectively.
[0037] FIG. 2B illustrates schematic, predicted structural
organizations of the spidroin proteins constructed according to
examples 5-8 (SEQ ID NOS: 9-13).
[0038] FIG. 3 is an alignment of C-terminal regions of MaSp1 and
MaSp2, illustrating their conserved nature (SEQ ID NOS: 24-55).
[0039] FIG. 4 illustrates macroscopic appearances of fibers formed
from spidroin proteins constructed according to examples 5-8. (A):
6Gly/Ala-CT.sub.hyb protein (SEQ ID NO: 13) fibers, bar 0.5 cm;
(B): 5Gly/Ala-CT.sub.nat (SEQ ID NO: 9) protein fibers, bar 1 cm.
(C): 5Gly/Ala-CT.sub.nat (SEQ ID NO: 9) protein fibers, bar 1
cm.
[0040] FIG. 5 shows scanning electron microscopy (SEM) micrographs
of fibers formed from spidroin proteins constructed according to
examples 5-8. Single fibers (a) and gel-phase (b, c) from
6Gly/Ala-CT.sub.hyb (SEQ ID NO: 13). Fibers of 5Gly/Ala-CT.sub.nat
(SEQ ID NO: 9), drawn in 75% methanol, air-dried and applied on
SEM-stubs (d, e, f). Fiber twisted before air-drying (e), end of
fiber (f).
[0041] FIG. 6 displays a circular dichroism (CD) spectrum of
6Gly/Ala-CT.sub.hyb (SEQ ID NO: 13) fiber.
[0042] FIG. 7 illustrates the results from a mouse mast cell
toxicity study, showing the numbers of live and dead cells after
three days of culture in the presence or absence of in vitro
produced silk fibers.
[0043] FIG. 8 is a picture of HEK293 cells following exposure to in
vitro produced silk fibers in a biocompatibility study.
[0044] FIG. 9 is a stress-strain curve displaying the tensile
strength of double drawn fibers from 5Gly/Ala-CT.sub.nat (SEQ ID
NO: 9).
[0045] FIG. 10 shows SEM micrographs of recombinant fibers from
5Gly/Ala-CT.sub.nat (SEQ ID NO: 9). a,b, Spontaneously formed
fibers. The close-up image (b) shows the fibrillar substructure.
The small fibril that bulges out (arrow) has a width of about 300
nm. c-f, Fibers after two stretching-relaxation cycles. c and d
shows the same fiber at different magnifications. e shows a cut
fiber end, and f shows a point of breakage after tensile
testing.
DETAILED DISCLOSURE OF THE INVENTION
[0046] The present invention is generally based on the
identification of a spidroin protein motif, which is sufficient for
recombinant production of spider silk fibers. The motif is based on
the deduced amino acid sequence from cloning and sequencing of a
partial major spidroin 1 (MaSp1) cDNA from Euprosthenops australis.
It follows that the isolated MaSp1 cDNA is useful as a starting
point for construction of novel spidroin genes, such as those
reported herein. The polymers which are formed from the proteins
resulting from the novel spidroin cDNAs are useful for their
physical properties, especially the useful combination of high
strength, elasticity and light weight. They are also useful for
their ability to support cell adherence and growth. The properties
of dragline silk are attractive in development of new materials for
medical or technical purposes. In particular, spider silks
according to the invention are useful in medical devices, such as
implants and medical products, such as wound closure systems,
band-aids, sutures, wound dressings, and scaffolds for tissue
engineering and guided cell regeneration. Spider silks according to
the invention are also particularly useful for use as textile or
fabric, such as in parachutes, bulletproof clothing, seat belts,
etc.
[0047] The term "fiber" as used herein relates to polymers having a
thickness of at least 1 .mu.m, preferably macroscopic polymers that
are visible to the human eye, i.e. having a thickness of at least 1
.mu.m, and have a considerable extension in length compared to its
thickness, preferably above 5 mm. The term "fiber" does not
encompass unstructured aggregates or precipitates.
[0048] The terms "major ampullate spidroin proteins", "spidroin
proteins" are used interchangeably throughout the description and
encompass all known major ampullate spidroin proteins, typically
abbreviated "MaSp", or "ADF" in the case of Araneus diadematus.
These major ampullate spidroin proteins are generally of two types,
1 and 2. These terms furthermore include the new proteins according
to the invention, as defined in the appended claims, and other
non-natural proteins with a high degree of identity and/or
similarity to the known major ampullate spidroin proteins.
[0049] The present inventors have utilized the identified spidroin
protein motif for construction of novel gene constructs, coding for
non-natural spidroin proteins. It has been found that a major
ampullate spidroin protein consisting of from 150 to 420 amino acid
residues, i.e. more than or equal to 150, preferably more than or
equal to 220, preferably more than or equal to 250, and less than
or equal to 420, preferably less than or equal to 380 amino acid
residues, preferably less than or equal to 320 amino acid residues,
preferably less than or equal to 280 amino acid residues, such as
220-360 amino acid residues, can be recombinantly produced, e.g. in
bacteria or other suitable production organisms. The resulting
spidroin proteins spontaneously form macroscopic silk fibers
according to the invention. This is a surprising result, since the
naturally occurring spidroin proteins and previously known,
recombinantly produced, fiber-forming spidroin proteins are
considerably longer than the proteins according to the invention.
Moreover, the naturally occurring spidroin proteins and previously
known, recombinantly produced, fiber-forming spidroin proteins tend
to contain a large number of internal repeats and require use of
spinning and/or harsh solvents for polymerization.
[0050] It is here for the first time shown that spidroin proteins
can spontaneously form fibers in vitro. The data presented herein
also show that only a fraction of the spidroin sequence need to be
present to dictate fiber formation. Moreover, a species hybrid
containing a Euprosthenops repetitive domain and a Nephila
non-repetitive C-terminal domain (c.f. Example 6C) forms fibers as
well, indicating that the fiber-forming potential of this motif is
robust.
[0051] In its general aspect, the major ampullate spidroin protein
according to the invention is defined by the formula REP-CT. The
REP protein fragment and the CT protein fragment are covalently
coupled, typically via a peptide bond.
[0052] The protein fragment REP has a repetitive character,
alternating between alanine-rich stretches and glycine-rich
stretches. The REP fragment generally contains more than 80, such
as more than 140, and less than 300, preferably less than 240, such
as less than 200, amino acid residues, and can itself be divided
into several L (linker) segments, A (alanine-rich) segments and G
(glycine-rich) segments, as will be explained in more detail below.
Typically, said linker segments, which are optional, are located at
the REP fragment terminals, while the remaining segments are in
turn alanine-rich and glycine-rich. Thus, the REP fragment can
generally have either of the following structures, wherein n is an
integer:
TABLE-US-00001 (SEQ ID NO: 17) L(AG).sub.nL, (SEQ ID NO: 56) such
as
LA.sub.1G.sub.1A.sub.2G.sub.2A.sub.3G.sub.3A.sub.4G.sub.4A.sub.5G-
.sub.5L; (SEQ ID NO: 18) L(AG).sub.nAL, (SEQ ID NO: 57) such as
LA.sub.1G.sub.1A.sub.2G.sub.2A.sub.3G.sub.3A.sub.4G.sub.4A.sub.5G-
.sub.5A.sub.6L; (SEQ ID NO: 19) L(GA).sub.nL, (SEQ ID NO: 58) such
as
LG.sub.1A.sub.1G.sub.2A.sub.2G.sub.3A.sub.3G.sub.4A.sub.4G.sub.5A-
.sub.5L; or (SEQ ID NO: 20) L(GA).sub.nGL, (SEQ ID NO: 59) such as
LG.sub.1A.sub.1G.sub.2A.sub.2G.sub.3A.sub.3G.sub.4A.sub.4G.sub.5A-
.sub.5G.sub.6L.
It follows that it is not critical whether an alanine-rich or a
glycine-rich segment is adjacent to the N-terminal or C-terminal
linker segments. It is preferred that n is an integer from 4 to 8,
more preferred from 4 to 6, i.e. n=4, n=5 or n=6.
[0053] In preferred embodiments, the alanine content of the REP
fragment according to the invention is above 20%, preferably above
25%, more preferably above 30%, and below 50%, preferably below
40%, more preferably below 35%. This is advantageous, since it is
contemplated that a higher alanine content provides a stiffer
and/or stronger and/or less extendible fiber. The reason for this
is likely to be that a higher alanine content is associated with a
higher content of .beta.-sheet structures in the fiber. Thus, in a
preferred embodiment, the .beta.-sheet content in a polymer, such
as a fiber, of the major ampullate spidroin protein according to
the invention is above 50%, i.e. more than 50% of the secondary
structure of the protein is in .beta.-sheet form.
[0054] In certain embodiments, the REP fragment is void of proline
residues, i.e. there are no Pro residues in the REP fragment.
[0055] Now turning to the segments that constitute the REP fragment
according to the invention, it shall be emphasized that each
segment is individual, i.e. any two A segments, any two G segments
or any two L segments of a specific REP fragment may be identical
or may not be identical. Thus, it is not a general feature of the
invention that each type of segment is identical within a specific
REP fragment. Rather, the following disclosure provides the skilled
person with guidelines how to design individual segments and gather
them into a REP fragment, which is a part of a functional spidroin
protein according to the invention.
[0056] It has been concluded from experimental data presented
herein that each individual A segment is an amino acid sequence
having from 8 to 18 amino acid residues. It is preferred that each
individual A segment contains from 13 to 15 amino acid residues. It
is also possible that a majority, or more than two, of the A
segments contain from 13 to 15 amino acid residues, and that a
minority, such as one or two, of the A segments contain from 8 to
18 amino acid residues, such as 8-12 or 16-18 amino acid residues.
A vast majority of these amino acid residues are alanine residues.
More specifically, from 0 to 3 of the amino acid residues are not
alanine residues, and the remaining amino acid residues are alanine
residues. Thus, all amino acid residues in each individual A
segment are alanine residues, with no exception or the exception of
one, two or three amino acid residues, which can be any amino acid.
It is preferred that the alanine-replacing amino acid(s) is (are)
natural amino acids, preferably individually selected from the
group of serine, glutamic acid, cysteine and glycine, more
preferably serine. Of course, it is possible that one or more of
the A segments are all-alanine segments, while the remaining A
segments contain 1-3 non-alanine residues, such as serine, glutamic
acid, cysteine or glycine.
[0057] In a preferred embodiment, each A segment contains 13-15
amino acid residues, including 10-15 alanine residues and 0-3
non-alanine residues as described above. In a more preferred
embodiment, each A segment contains 13-15 amino acid residues,
including 12-15 alanine residues and 0-1 non-alanine residues as
described above.
[0058] It is preferred that each individual A segment has at least
80% identity to an amino acid sequence selected from the group of
amino acid residues 7-19, 43-56, 71-83, 107-120, 135-147, 171-183,
198-211, 235-248, 266-279, 294-306, 330-342, 357-370, 394-406,
421-434, 458-470, 489-502, 517-529, 553-566, 581-594, 618-630,
648-661, 676-688, 712-725, 740-752, 776-789, 804-816, 840-853,
868-880, 904-917, 932-945, 969-981, 999-1013, 1028-1042 and
1060-1073 of SEQ ID NO: 3. Each sequence of this group corresponds
to a segment of the naturally occurring sequence of Euprosthenops
australis MaSp1 protein, which is deduced from cloning of the
corresponding cDNA, see Examples 1-2 and FIG. 1-2A. Alternatively,
each individual A segment has at least 80% identity to an amino
acid sequence selected from the group of amino acid residues 31-42,
61-75, 90-104, 122-135 and 153-171 of SEQ ID NO: 9, amino acid
residues 12-25, 46-60, 75-88, 112-119, 150-158 and 173-180 of SEQ
ID NO: 13, amino acid residues 31-42 of SEQ ID NO: 14 and amino
acid residues 122-135 of SEQ ID NO: 15. Each sequence of this group
corresponds to a segment of expressed, non-natural spidroin
proteins according to the invention, which proteins have capacity
to form silk fibers under appropriate conditions. See Examples 5-8,
12 and FIG. 2B. Without wishing to be bound by any particular
theory, it is envisaged that A segments according to the invention
form helical structures or beta sheets.
[0059] The term "% identity", as used throughout the specification
and the appended claims, is calculated as follows. The query
sequence is aligned to the target sequence using the CLUSTAL W
algorithm (Thompson, J. D., Higgins, D. G. and Gibson, T. J.,
Nucleic Acids Research, 22: 4673-4680 (1994)). The amino acid
residues at each position are compared, and the percentage of
positions in the query sequence that have identical correspondences
in the target sequence is reported as % identity.
[0060] The term "% similarity", as used throughout the
specification and the appended claims, is calculated as described
for "% identity", with the exception that the hydrophobic residues
Ala, Val, Phe, Pro, Leu, Ile, Trp, Met and Cys are similar; the
basic residues Lys, Arg and His are similar; the acidic residues
Glu and Asp are similar; and the hydrophilic, uncharged residues
Gln, Asn, Ser, Thr and Tyr are similar. The remaining natural amino
acid Gly is not similar to any other amino acid in this
context.
[0061] Throughout this description, alternative embodiments
according to the invention fulfill, instead of the specified
percentage of identity, the corresponding percentage of similarity.
Other alternative embodiments fulfill the specified percentage of
identity as well as another, higher percentage of similarity,
selected from the group of preferred percentages of identity for
each sequence. For example, a sequence may be 70% similar to
another sequence; or it may be 70% identical to another sequence;
or it may be 70% identical and 90% similar to another sequence.
[0062] In preferred embodiments according to the invention, each
individual A segment has at least 90%, more preferably 95%, most
preferably 100%, identity to an amino acid sequence selected from
the group of amino acid residues 7-19, 43-56, 71-83, 107-120,
135-147, 171-183, 198-211, 235-248, 266-279, 294-306, 330-342,
357-370, 394-406, 421-434, 458-470, 489-502, 517-529, 553-566,
581-594, 618-630, 648-661, 676-688, 712-725, 740-752, 776-789,
804-816, 840-853, 868-880, 904-917, 932-945, 969-981, 999-1013,
1028-1042 and 1060-1073 of SEQ ID NO: 3; amino acid residues 31-42,
61-75, 90-104, 122-135 and 153-171 of SEQ ID NO: 9; amino acid
residues 12-25, 46-60, 75-88, 112-119, 150-158 and 173-180 of SEQ
ID NO: 13; amino acid residues 31-42 of SEQ ID NO: 14; and amino
acid residues 122-135 of SEQ ID NO: 15. Thus, in certain
embodiments according to the invention, each individual A segment
is identical to an amino acid sequence selected from the
above-mentioned amino acid segments.
[0063] Furthermore, it has been concluded from experimental data
presented herein that each individual G segment is an amino acid
sequence of from 12 to 30 amino acid residues. It is preferred that
each individual G segment consists of from 14 to 23 amino acid
residues. At least 40% of the amino acid residues of each G segment
are glycine residues. Typically the glycine content of each
individual G segment is in the range of 40-60%.
[0064] It is preferred that each individual G segment has at least
80% identity to an amino acid sequence selected from the group of
amino acid residues 20-42, 57-70, 84-106, 121-134, 148-170,
184-197, 212-234, 249-265, 280-293, 307-329, 343-356, 371-393,
407-420, 435-457, 471-488, 503-516, 530-552, 567-580, 595-617,
631-647, 662-675, 689-711, 726-739, 753-775, 790-803, 817-839,
854-867, 881-903, 918-931, 946-968, 982-998, 1014-1027, 1043-1059
and 1074-1092 of SEQ ID NO: 3. Each sequence of this group
corresponds to a segment of the naturally occurring sequence of
Euprosthenops australis MaSp1 protein, which is deduced from
cloning of the corresponding cDNA, see Examples 1-2 and FIG. 1-2A.
Alternatively, each individual G segment has at least 80% identity
to an amino acid sequence selected from the group of amino acid
residues 11-30, 43-60, 76-89, 105-121 and 136-152 of SEQ ID NO: 9
and amino acid residues 1-11, 26-45, 61-74, 89-111, 120-149 and
159-172 of SEQ ID NO: 13. Each sequence of this group corresponds
to a segment of expressed, non-natural spidroin proteins according
to the invention, which proteins have capacity to form silk fibers
under appropriate conditions. See Examples 5-8, 12 and FIG. 2B.
[0065] In preferred embodiments according to the invention, each
individual G segment has at least 90%, more preferably 95%, most
preferably 100%, identity to an amino acid sequence selected from
the group of amino acid residues 20-42, 57-70, 84-106, 121-134,
148-170, 184-197, 212-234, 249-265, 280-293, 307-329, 343-356,
371-393, 407-420, 435-457, 471-488, 503-516, 530-552, 567-580,
595-617, 631-647, 662-675, 689-711, 726-739, 753-775, 790-803,
817-839, 854-867, 881-903, 918-931, 946-968, 982-998, 1014-1027,
1043-1059 and 1074-1092 of SEQ ID NO: 3; amino acid residues 11-30,
43-60, 76-89, 105-121 and 136-152 of SEQ ID NO: 9; and amino acid
residues 1-11, 26-45, 61-74, 89-111, 120-149 and 159-172 of SEQ ID
NO: 13. Thus, in certain embodiments according to the invention,
each individual G segment is identical to an amino acid sequence
selected from the above-mentioned amino acid segments.
[0066] In certain embodiments, the first two amino acid residues of
each G segment according to the invention are not -Gln-Gln-.
[0067] In certain embodiments, the position corresponding to the
conserved Tyr residue (i.e. corresponding to position 16 in SEQ ID
NO: 5, position 10 in SEQ ID NO: 6 and position 7 in SEQ ID NO: 7)
is not Phe in any G segment according to the invention.
[0068] In certain embodiments, the position corresponding to the
conserved Tyr residue (i.e. corresponding to position 16 in SEQ ID
NO: 5, position 10 in SEQ ID NO: 6 and position 7 in SEQ ID NO: 7)
is Tyr in each G segment according to the invention.
[0069] It follows that certain embodiments of the proteins
according to the invention display a combination of the
above-mentioned limitations, i.e. the first two amino acid residues
of each G segment according to the invention are not -Gln-Gln-, and
the conserved Tyr residue (i.e. corresponding to position 16 in SEQ
ID NO: 5, position 10 in SEQ ID NO: 6 and position 7 in SEQ ID NO:
7) is Tyr in each G segment according to the invention. In certain
embodiments, the above-mentioned limitations, taken separately or
in any possible combination, can be further combined with the
limitation that the REP fragment is void of proline residues, as
discussed hereinabove.
[0070] With reference to FIGS. 1-2 and Examples 3-4, there are the
three subtypes of the G segment according to the invention. This
classification is based upon careful analysis of the Euprosthenops
australis MaSp1 protein sequence (FIGS. 1-2A), and the information
has been employed and verified in the construction of novel,
non-natural spider silk proteins (FIG. 2B).
[0071] The first subtype of the G segment according to the
invention is represented by the amino acid one letter consensus
sequence GQG(G/S)QGG(Q/Y)GG (L/Q)GQGGYGQGA GSS, as shown in FIG. 2A
and SEQ ID NO: 5. This first, and generally the longest, G segment
subtype typically contains 23 amino acid residues, but may contain
as little as 17 amino acid residues, and lacks charged residues or
contain one charged residue. Thus, it is preferred that this first
G segment subtype contains 17-23 amino acid residues, but it is
contemplated that it may contain as few as 12 or as many as 30
amino acid residues. Without wishing to be bound by any particular
theory, it is envisaged that this subtype forms coil structures or
3.sub.1-helix structures. Representative G segments of this first
subtype are amino acid residues 20-42, 84-106, 148-170, 212-234,
307-329, 371-393, 435-457, 530-552, 595-617, 689-711, 753-775,
817-839, 881-903, 946-968, 1043-1059 and 1074-1092 of SEQ ID NO: 3;
amino acid residues 11-30, 105-121 and 136-152 of SEQ ID NO: 9; and
amino acid residues 26-45 and 89-111 of SEQ ID NO: 13. Alternative
G segments of this first subtype are amino acid residues 120-149
and 159-172 of SEQ ID NO: 13. In certain embodiments, the first two
amino acid residues of each G segment of this first subtype
according to the invention are not -Gln-Gln-.
[0072] The second subtype of the G segment according to the
invention is represented by the amino acid one letter consensus
sequence GQGGQGQG(G/R)Y GQG(A/S)G(S/G)S, as shown in FIG. 2A and
SEQ ID NO: 6. This second, generally mid-sized, G segment subtype
typically contains 17 amino acid residues and lacks charged
residues or contain one charged residue. It is preferred that this
second G segment subtype contains 14-20 amino acid residues, but it
is contemplated that it may contain as few as 12 or as many as 30
amino acid residues. Without wishing to be bound by any particular
theory, it is envisaged that this subtype forms coil structures.
Representative G segments of this second subtype are amino acid
residues 249-265, 471-488, 631-647 and 982-998 of SEQ ID NO: 3; and
amino acid residues 43-60 of SEQ ID NO: 9.
[0073] The third subtype of the G segment according to the
invention is represented by the amino acid one letter consensus
sequence G(R/Q)GQG(G/R)YGQG (A/S/V)GGN, as shown in FIG. 2A and SEQ
ID NO: 7. This third G segment subtype typically contains 14 amino
acid residues, and is generally the shortest of the G segment
subtypes according to the invention. It is preferred that this
third G segment subtype contains 12-17 amino acid residues, but it
is contemplated that it may contain as many as 23 amino acid
residues. Without wishing to be bound by any particular theory, it
is envisaged that this subtype forms turn structures.
Representative G segments of this third subtype are amino acid
residues 57-70, 121-134, 184-197, 280-293, 343-356, 407-420,
503-516, 567-580, 662-675, 726-739, 790-803, 854-867, 918-931,
1014-1027 of SEQ ID NO: 3; amino acid residues 76-89 of SEQ ID NO:
9; and amino acid residues 61-74 of SEQ ID NO: 13. An alternative G
segment of this third subtype is amino acid residues 1-11 of SEQ ID
NO: 13.
[0074] Thus, in preferred embodiments, each individual G segment
has at least 80%, preferably 90%, more preferably 95%, identity to
an amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 6 and
SEQ ID NO: 7.
[0075] In a preferred embodiment of the alternating sequence of A
and G segments of the REP fragment, every second G segment is of
the first subtype, while the remaining G segments are of the third
subtype, e.g.
TABLE-US-00002 (SEQ ID NO: 56) . . .
A.sub.1G.sub.shortA.sub.2G.sub.longA.sub.3G.sub.shortA.sub.4G.sub.l-
ongA.sub.5G.sub.short . . . In
another preferred embodiment of the REP fragment, one G segment of
the second subtype interrupts the G segment regularity via an
insertion, e.g.
TABLE-US-00003 (SEQ ID NO: 56) . . .
A.sub.1G.sub.shortA.sub.2G.sub.longA.sub.3G.sub.midA.sub.4G.sub.sho-
rtA.sub.5G.sub.long . . .
[0076] Each individual L segment represents an optional linker
amino acid sequence, which may contain from 0 to 20 amino acid
residues, such as from 0 to 10 amino acid residues. While this
segment is optional and not functionally critical for the spidroin
protein, its presence still allows for fully functional spidroin
proteins, forming spider silk fibers according to the invention.
There are also linker amino acid sequences present in the
repetitive part (SEQ ID NO: 3) of the deduced amino acid sequence
of the MaSp1 protein from Euprosthenops australis. In particular,
the amino acid sequence of a linker segment may resemble any of the
described A or G segments, but usually not sufficiently to meet
their criteria as defined herein.
[0077] As shown in FIG. 2A, a linker segment arranged at the
C-terminal part of the REP fragment can be represented by the amino
acid one letter consensus sequences ASASAAASAA STVANSVS (SEQ ID NO:
60) and ASAASAAA (SEQ ID NO: 61), which are rich in alanine. In
fact, the second sequence can be considered to be an A segment
according to the invention, while the first sequence has a high
degree of similarity to A segments according to the invention.
Another example of a linker segment according the invention has the
one letter amino acid sequence GSAMGQGS (SEQ ID NO: 62), which is
rich in glycine and has a high degree of similarity to G segments
according to the invention.
[0078] Representative L segments are amino acid residues 1-6 and
1093-1110 of SEQ ID NO: 3; amino acid residues 1-10 and 153-171 of
SEQ ID NO: 9; and amino acid residues 173-180 of SEQ ID NO: 13, but
the skilled person in the art will readily recognize that there are
many suitable alternative amino acid sequences for these segments.
In one embodiment of the REP fragment according to the invention,
one of the L segments contains 0 amino acids, i.e. one of the L
segments is void. In another embodiment of the REP fragment
according to the invention, both L segments contain 0 amino acids,
i.e. both L segments are void. Thus, these embodiments of the REP
fragments according to the invention may be schematically
represented as follows: (AG).sub.nL, (AG).sub.nAL, (GA).sub.nL,
(GA).sub.nGL; L(AG).sub.n, L(AG).sub.nA, L(GA).sub.n, L(GA).sub.nG;
and (AG).sub.n, (AG).sub.nA, (GA).sub.n, (GA).sub.nG. Any of these
REP fragments are suitable for use with any CT fragment as defined
below.
[0079] The C-terminal (CT) fragment of the spidroin protein
according to the invention has a high degree of similarity to the
C-terminal amino acid sequence of spidroin proteins. As shown in
FIG. 3, this amino acid sequence is well conserved among various
species and spidroin proteins, including MaSp1 and MaSp2. It is
demonstrated in the following examples that it is not critical
which specific CT fragment is present in spidroin proteins
according to the invention, as long as the CT fragment is not
entirely missing. Thus, the CT fragment according to the invention
can be selected from any of the amino acid sequences shown in FIG.
3 or sequences with a high degree of similarity. It is notable that
the CT.sub.hyb fragment of SEQ ID NO: 13 has 96% identity to the
consensus amino acid sequence SEQ ID NO: 8, while the CT.sub.nat
fragment of SEQ ID NO: 9 displays only 59% identity to the
consensus amino acid sequence SEQ ID NO: 8. This illustrates that a
wide variety of C-terminal sequences can be used in the spidroin
protein according to the invention.
[0080] The sequence of the CT fragment according to the invention
has at least 50% identity, preferably at least 60% identity, to the
consensus amino acid sequence SEQ ID NO: 8, which is based on the
amino acid sequences of FIG. 3. In a preferred embodiment, the
sequence of the CT fragment according to the invention has at least
65% identity, preferably at least 70% identity, to amino acid
residues 1-71 of the consensus amino acid sequence SEQ ID NO: 8. In
preferred embodiments, the CT fragment according to the invention
has furthermore 70%, preferably 80%, similarity to the consensus
amino acid sequence SEQ ID NO: 8, or amino acid residues 1-71
thereof.
[0081] Representative CT fragments according to the invention are
the Euprosthenops australis sequence SEQ ID NO: 4, the
Euprosthenops australis-derived amino acid residues 172-269 of SEQ
ID NO: 9 and amino acid residues 181-276 of SEQ ID NO: 13, alleged
to be derived from Euprosthenops sp (Pouchkina-Stantcheva, N. N.
& McQueen-Mason, S. J. Molecular studies of a novel dragline
silk from a nursery web spider, Euprosthenops sp. (Pisauridae).
Comp Biochem Physiol B Biochem Mol Biol 138, 371-376 (2004)), but
with a high degree of similarity to MaSp1 from Nephila clavipes and
Nephila senegalensis. Thus, according to a preferred aspect of the
invention, the CT fragment has at least 80% identity to SEQ ID NO:
4, amino acid residues 172-269 of SEQ ID NO: 9, amino acid residues
181-276 of SEQ ID NO: 13, amino acid residues 172-269 of SEQ ID NO:
16 or any individual MaSp1/ADF-4 amino acid sequence of FIG. 3 and
Example 4. In preferred aspects of the invention, the CT fragment
has at least 90%, such as at least 95% identity, to SEQ ID NO: 4,
amino acid residues 172-269 of SEQ ID NO: 9, amino acid residues
181-276 of SEQ ID NO: 13, amino acid residues 172-269 of SEQ ID NO:
16 or any individual MaSp1/ADF-4 amino acid sequence of FIG. 3 and
Example 4. In preferred aspects of the invention, the CT fragment
is identical to SEQ ID NO: 4, amino acid residues 172-269 of SEQ ID
NO: 9, amino acid residues 181-276 of SEQ ID NO: 13, amino acid
residues 172-269 of SEQ ID NO: 16 or any individual MaSp1/ADF-4
amino acid sequence of FIG. 3 and Example 4.
[0082] The CT fragment typically consists of from 70 to 120 amino
acid residues. It is preferred that the CT fragment contains at
least 70, or more than 80, preferably more than 90, amino acid
residues. It is also preferred that the CT fragment contains at
most 120, or less than 110 amino acid residues. A typical CT
fragment contains approximately 100 amino acid residues.
[0083] According to another aspect, the present invention provides
an isolated fusion protein consisting of a first protein fragment,
which is a major ampullate spidroin protein, preferably consisting
of from 150 to 420 amino acid residues, and a second protein
fragment, which comprises a fusion partner and a cleavage agent
recognition site. The first protein fragment is coupled via the
cleavage agent recognition site to the fusion partner, i.e. the
fusion partner can be cleaved off by treating the fusion protein
with a suitable cleaving agent under appropriate conditions,
providing a major ampullate spidroin protein, preferably consisting
of from 150 to 420 amino acid residues. An advantage with this
fusion protein is that large amounts thereof can be produced in
solution, preferably in a physiological medium, typically a
buffered aqueous medium, such as a 10-100 mM Tris-HCl buffer, pH
6-9, without causing precipitation and other production problems
when produced in suitable hosts, such as bacteria, preferably E.
coli. The fusion proteins in the solution are soluble for long time
periods, typically days or weeks, which facilitates large-scale
production and decreases the risk for protein aggregation. By the
terms "soluble" and "in solution" is meant that the protein is not
visibly aggregated and does not precipitate from the solvent at 60
000.times.g. At wish, the fusion proteins in the solution can be
subjected to cleavage using a suitable cleaving agent, providing a
major ampullate spidroin protein which spontaneously forms silk
fibers.
[0084] In a preferred aspect, the present invention provides an
isolated fusion protein selected from the group of X-REP-CT and
REP-CT-X, preferably X-REP-CT. REP and CT are protein fragments
according to the invention, implying that the resulting MaSp
protein of the form REP-CT is a MaSp protein according to the
invention. X is a protein fragment comprising a fusion partner and
a cleavage agent recognition site as defined above. The combined
protein fragment REP-CT is coupled via the cleavage agent
recognition site to the fusion partner.
[0085] Fusion partners according to the invention include any
protein fragment which improves the solubility and/or stability of
its partner protein fragment, here the MaSp protein according to
the invention. The fusion partner also provides a suitable handle
for affinity purification. Without being limited thereto, examples
of fusion partners according to the invention include thioredoxin,
maltose-binding protein, glutathione S-transferase (GST), MTB32-C,
Gb1, ZZ and Nus A. The skilled person is well aware of alternative
suitable fusion partners. In a preferred embodiment of the
invention, the fusion partner is a thioredoxin moiety (ThrX) in
combination with a His tag and an S tag. In one preferred
embodiment of the invention, the fusion partner is a ThrX moiety in
combination with two His tags, i.e. His-tag/ThrX/His-tag. In
another preferred embodiment of the invention, the fusion partner
is a thioredoxin moiety (ThrX).
[0086] The cleavage agent recognition site is situated at that X
protein fragment terminal which is coupled to the MaSp protein
fragment, so that cleavage at the recognition site results in a
MaSp protein and a fusion partner. Without being limited thereto,
examples of the cleavage agent recognition site according to the
invention include a thrombin recognition site having the amino acid
sequence LVPRGS (SEQ ID NO: 63) (cleaves between R and G); an
enterokinase recognition site having the amino acid sequence DDDK
(SEQ ID NO: 64) (cleaves after K); an hydroxylamine recognition
site having the amino acid sequence NG (cleaves between N and G); a
HRV 3C protease recognition site having the amino acid sequence
LGVLFQGP (SEQ ID NO: 65) (cleaves between Q and G); a Factor Xa
recognition site having the amino acid sequence I(E/D)GR (SEQ ID
NO: 66) (cleaves after R); a TEV recognition site having the amino
acid sequence EXXYXQ(G/S) (SEQ ID NO: 67), commonly ENLYFQG (SEQ ID
NO: 68) (cleaves between Q and G/S), an Ac-TEV recognition site
having the amino acid sequence EDNLYFQG (SEQ ID NO: 69)(cleaves
between Q and G); and a PreScission recognition site having the
amino acid sequence LEVLFQGP (SEQ ID NO: 70) (cleaves between Q and
G). Other suitable recognition sites are the cleavage sites for
trypsin, endoproteinase, V8 protease, pepsin and CNBr. Further
examples of suitable cleavage recognition sites are well within the
reach of the skilled person. In a preferred embodiment of the
invention, the cleavage agent recognition site is a thrombin
recognition site.
[0087] In a preferred embodiment, the X fragment according to the
invention has the structure ThrX/His-tag/S-tag/thrombin cleavage
site, and the X fragment is coupled to the N-terminal of the REP-CT
protein fragment according to the invention.
[0088] In one preferred embodiment, the X fragment according to the
invention has the structure His-tag/ThrX/His-tag/thrombin cleavage
site, and the X fragment is coupled to the N-terminal of the REP-CT
protein fragment according to the invention.
[0089] According to another aspect, the present invention provides
a method of producing a major ampullate spidroin protein according
to the invention. In the first step, a solution of a fusion protein
according to the invention in a liquid medium is provided.
Preferably, the fusion protein does not aggregate, and therefore,
resolubilization procedures are not required. The fusion protein
can be recombinantly produced and purified using an affinity handle
at the fusion protein, such as a His-tag or any suitable epitope in
the fusion protein. The liquid medium can be any suitable medium,
preferably a physiological medium, typically a buffered aqueous
medium, such as a 10-100 mM Tris-HCl buffer, pH 6-9. In the second
step, a cleavage agent according to the invention is added to the
liquid medium in order to achieve cleavage of the fusion protein at
the cleavage agent recognition site. As disclosed above, the major
ampullate spidroin protein according to the invention is thereby
obtained. In a third, optional step, the thus obtained major
ampullate spidroin protein is isolated from the liquid medium using
suitable isolation techniques, such as chromatography and/or
filtration.
[0090] According to yet another aspect, the present invention
provides a method of producing a polymer of a major ampullate
spidroin protein according to the invention. In the first step, a
solution of a fusion protein according to the invention in a liquid
medium is provided. Preferably, the fusion protein does not
aggregate, and therefore, resolubilization procedures are not
required. The fusion protein can be recombinantly produced and
purified using an affinity handle at the fusion protein, such as a
His-tag or any suitable epitope in the fusion protein. The liquid
medium can be any suitable medium, preferably a physiological
medium, typically a buffered aqueous medium, such as a 10-100 mM
Tris-HCl buffer, pH 6-9. In the second step, a cleavage agent
according to the invention is added to the liquid medium in order
to achieve cleavage of the fusion protein at the cleavage agent
recognition site. As disclosed above, the major ampullate spidroin
protein according to the invention is thereby obtained. In the
third step, the thus obtained major ampullate spidroin protein is
allowed to polymerize in the liquid medium. The polymerization
typically initiates at the interface between two different phases,
such as liquid/air, liquid/solid, and water/oil interfaces. Thus,
this third step may also further comprise providing an interface
between the liquid medium and another phase. The other phase is
selected from the group consisting of a gas phase, a liquid phase
and a solid phase. As detailed above, the liquid medium is
typically an aqueous medium, and suitable other phases are for
instance air and water-immiscible organic solvents, such as oil,
e.g. mineral oil suitable for PCR reactions. The presence of the
resulting interface stimulates polymerization at the interface or
in the region surrounding the interface, which region extends into
the liquid medium, such that said polymerizing initiates at said
interface or in said interface region. Preferred interfaces include
water/air and water/oil interfaces. Polymerization typically occurs
spontaneously within minutes or a few hours, such as within from 1
min to 5 h, upon incubation at room temperature. In a fourth,
optional step, the thus obtained polymer of the major ampullate
spidroin protein is isolated from the liquid medium using suitable
isolation techniques.
[0091] As discussed above, fiber formation is induced by
proteolytic release of the miniature spidroin from the fusion
protein. If the cleavage reaction is performed in a tube that is
gently wagged from side to side, a fiber is formed at the air-water
interface along the tube. The tube can be made of any suitable
material, such as plastic or glass. If the cleavage mixture is
allowed to stand still, a film is formed at the air-water
interface. If oil is added on top of the aqueous cleavage mixture,
a film is formed at the oil-water interface, either if allowed to
stand still or if wagged. If the cleavage mixture is foamed, e.g.
by bubbling of air or whipping, the foam is stable and solidifies
if allowed to dry.
[0092] Using the method(s) of the present invention, it is possible
to recombinantly produce large amounts of fusion proteins according
the invention, which can be cleaved and allowed to polymerize at
desire. This provides a better control of the polymerization
process and allows for optimization of parameters for obtaining
silk fibers with desirable properties.
[0093] The major ampullate spidroin protein according to the
invention is typically recombinantly produced using a variety of
suitable hosts. According to another aspect, the present invention
therefore provides an isolated polynucleic acid molecule comprising
a nucleic acid sequence which encodes a major ampullate spidroin
protein according to the invention, or its complementary nucleic
acid sequence, such as SEQ ID NOS: 9-13, preferably SEQ ID NOS: 9,
12 and 13. These polynucleic acid molecules as well as polynucleic
acid molecules coding for the various proteins disclosed herein
(SEQ ID NOS: 2-16) may also be useful in further developments of
non-natural spidroin proteins or production systems therefor.
[0094] The fusion protein according to the invention is typically
recombinantly produced using a variety of suitable hosts, such as
bacteria, yeast, mammalian cells, plants, insect cells, and
transgenic animals. It is preferred that the fusion protein
according to the invention is produced in bacteria.
[0095] According to another aspect, the present invention therefore
provides an isolated polynucleic acid molecule comprising a nucleic
acid sequence which encodes a fusion protein according to the
invention, or its complementary nucleic acid sequence. The
polynucleic acid molecule may also be useful in further
developments of non-natural spidroin proteins or production systems
therefor.
[0096] Polynucleic acid molecules according to the invention can be
DNA molecules, including cDNA molecules, or RNA molecules. As the
skilled person is well aware, a nucleic acid sequence may as well
be described by its complementary nucleic acid sequence. Therefore,
nucleic acid sequences that are complementary to the nucleic acid
sequences according to the invention are also encompassed by the
protective scope of the invention.
[0097] According to one aspect, the present invention provides a
method of producing a soluble fusion protein according to the
invention. In the first step, a polynucleic acid molecule which
encodes a fusion protein according to the invention is expressed in
a suitable host. In the second step, the thus obtained soluble
fusion protein in step is isolated, e.g. using chromatography
and/or filtration. Optionally, said second step of isolating the
soluble fusion protein involves removal of LPS and other
pyrogens.
[0098] The present invention further provides a method of producing
a major ampullate spidroin protein according to the invention. In
the first step, a polynucleic acid molecule which encodes a fusion
protein according to the invention is expressed in a suitable host.
In the second step, the thus obtained soluble fusion protein is
isolated, e.g. using chromatography and/or filtration. In the third
step, a solution of the isolated fusion protein is provided, and in
the fourth step, a suitable cleaving agent is added to the liquid
medium. This achieves cleavage of the fusion protein at the
cleavage agent recognition site, and thereby provides the major
ampullate spidroin protein. In an optional fifth step, the thus
obtained major ampullate spidroin protein is isolated from the
liquid medium. Further optionally, said second step of isolating
the soluble fusion protein, and optionally the fifth step of
isolating the major ampullate spidroin protein, involve(s) removal
of LPS and other pyrogens.
[0099] The present invention also provides a method of producing a
polymer of a major ampullate spidroin protein according to the
invention. In the first step, a polynucleic acid molecule which
encodes a fusion protein according to the invention is expressed in
a suitable host. In the second step, the thus obtained soluble
fusion protein is isolated, e.g. using chromatography and/or
filtration. In the third step, a solution of the isolated fusion
protein is provided, and in the fourth step, a suitable cleaving
agent is added to the liquid medium. This achieves cleavage of the
fusion protein at the cleavage agent recognition site, and thereby
provides the major ampullate spidroin protein. In the fifth step,
the thus obtained major ampullate spidroin protein is allowed to
polymerize in the liquid medium. The polymerization typically
initiates at the interface between two different phases, such as
liquid/air, liquid/solid, and water/oil interfaces. Thus, this
fifth step may also further comprise providing an interface between
the liquid medium and another phase. The other phase is selected
from the group consisting of a gas phase, a liquid phase and a
solid phase. As detailed above, the liquid medium is typically an
aqueous medium, and suitable other phases are for instance air and
water-immiscible organic solvents, such as oil, e.g. mineral oil
suitable for PCR reactions. The presence of the resulting interface
stimulates polymerization at the interface or in the region
surrounding the interface, which region extends into the liquid
medium, such that said polymerizing initiates at said interface or
in said interface region. Preferred interfaces include water/air
and water/oil interfaces. Polymerization typically occurs
spontaneously within minutes or a few hours, such as within from 1
min to 5 h, upon incubation at room temperature. In an optional
sixth step, the thus obtained polymer is isolated from the liquid
medium.
[0100] In order to obtain a protein with low pyrogenic content,
which is an obligate for usage as a biomaterial in vivo, a
purification protocol optimized for removal of lipopolysaccharides
(LPS) has been developed. To avoid contamination by released LPS,
the producing bacterial cells are subjected to washing steps with
altering CaCl.sub.2 and EDTA. After cell lysis, all subsequent
purifications steps are performed in low conductivity buffers in
order to minimize hydrophobic interactions between the target
protein and LPS. The LPS content is further minimized by passage of
the protein solution through an Endotrap column, which has a ligand
that specifically adsorbs LPS. To assure constant low content of
LPS and other pyrogens, all batches are analyzed using an in vitro
pyrogen test (IPT) and/or a Limulus amebocyte lysate (LAL) kinetic
assay. Although produced in a gram-negative bacterial host, the
recombinant spidroin proteins can be purified so that residual
levels of LPS and other pyrogens are below the limits required for
animal tests, i.e. below 25 EU/implant. In certain embodiments
according to the invention, the content of LPS and other pyrogens
in the isolated fusion protein is 1 EU/mg protein or lower. In
certain embodiments according to the invention, the content of LPS
and other pyrogens in the isolated major ampullate spidroin protein
is 1 EU/mg protein or lower, preferably 0.25 EU/mg protein or
lower.
[0101] According to another aspect, the present invention provides
a polymer of a major ampullate spidroin protein according to the
invention. In a preferred embodiment, the polymer of this protein
is obtainable by any one of the methods therefor according to the
invention.
[0102] In preferred embodiments, the .beta.-sheet content of the
polymer of the major ampullate spidroin protein according to the
invention is above 50%, i.e. more than 50% of the secondary
structure of the polymer of this protein is in .beta.-sheet form.
This is advantageous, since it is contemplated that a higher
content of .beta.-sheet structures provides a stiffer and/or
stronger and/or less extendible fiber.
[0103] It is preferable that the polymer of the spidroin protein
according to the invention is a fiber with a macroscopic size, i.e.
with a diameter above 1 .mu.m, preferably above 10 .mu.m and a
length above 5 mm. It is preferred that the fiber has a diameter in
the range of 10-400 .mu.m, preferably 60-120 .mu.m, and a length in
the range of 0.5-300 cm, preferably 1-100 cm. Other preferred
ranges are 0.5-30 cm and 1-20 cm. It is also preferred that the
polymer of the spidroin protein according to the invention has a
tensile strength above 1 MPa, preferably above 2 MPa, more
preferably 10 MPa or higher. It is preferred that the polymer of
the spidroin protein according to the invention has a tensile
strength above 100 MPa, more preferably 200 MPa or higher. The
fiber has the capacity to remain intact during physical
manipulation, i.e. can be used for spinning, weaving, twisting,
crocheting and similar procedures.
[0104] In other preferred embodiments, the polymer of the spidroin
protein according to the invention forms a foam, a gel, a mesh or a
film.
[0105] According to yet another aspect, the present invention
provides a novel use of a protein fragment comprising a fusion
partner and a cleavage agent recognition site for the manufacture
of a fusion protein. The fusion protein is comprising said protein
fragment and a spider silk protein fragment according to the
invention, and the two fragments are coupled via said cleavage
agent recognition site. The spider silk protein fragment preferably
consists of from 150 to 420 amino acid residues.
[0106] The present invention will in the following be further
illustrated by the following non-limiting examples.
EXAMPLES
Example 1
Cloning and Sequencing of Euprosthenops australis MaSp1 cDNA
[0107] The major ampullate glands from approximately 100 adult
female Euprosthenops australis spiders, collected in South Africa,
were used to construct a custom-made pDONR222-based CloneMiner cDNA
library (Invitrogen, Paisley, UK). cDNA clones encoding the MaSp1
protein were obtained by screening the library with a cDNA probe
encoding an alanine- and glycine-rich fragment originating from
Euprosthenops spiders of unknown subspecies. Colony blotting and
detection were performed with an ECL direct labelling and detection
system (Amersham Biosciences, Uppsala, Sweden) according to the
manufacturer's instruction.
[0108] One single clone was chosen for further characterization. To
obtain full length sequence of the cDNA insert from this clone,
nested deletions were made using the Erase-a-Base System (Promega,
Southampton, UK), and sequencing was performed on a MegaBase 1000
instrument (Amersham Biosciences).
[0109] The resulting 3.8 kb cDNA (SEQ ID NO: 1) encodes a MaSp1
protein (SEQ ID NO: 2) of 1207 amino acid residues, containing a
repetitive fragment of 34 alanine- and glycine-rich segments (SEQ
ID NO: 3), and a C-terminal non-repetitive fragment of 97 amino
acid residues (SEQ ID NO: 4).
Example 2
Sequence Analysis of the Repetitive Fragment of Euprosthenops
australis MaSp1 Protein
[0110] The repetitive fragment of the Euprosthenops australis MaSp1
protein sequence of Example 1 (SEQ ID NO: 3) was further analyzed
by alignment of the repetitive segments of the fragment, see FIG.
1. The alignment was carefully scrutinized and the following
structural information was concluded.
[0111] The alanine-rich segments of the Euprosthenops australis
MaSp1 protein are 13-15 amino acid residues long and consists of
only alanine residues or all alanine residues but one residue,
which is a serine, glutamate or glycine residue.
[0112] The repetitive fragment of the Euprosthenops australis MaSp1
protein further contains three related, but distinct, types of
glycine-rich segments, c.f. FIG. 2A. Two of the glycine-rich
segments differ almost only in length and occurrence; the most
common glycine-rich segment contains 23 amino acid residues, while
a less abundant variant contains 17 amino acid residues. Both of
these glycine-rich segments generally lack charged residues or
contain one charged residue. In contrast, the shortest glycine-rich
segment, containing 14 amino acid residues, uniquely contains the
sequence GRGQG (SEQ ID NO: 71) or GQGQG (SEQ ID NO: 72) at the
N-terminal end, and GN at the C-terminal end.
[0113] The longest glycine-rich segment is represented by the amino
acid one letter consensus sequence GQG(G/S)QGG(Q/Y)GG
(L/Q)GQGGYGQGA GSS (SEQ ID NO: 5), and lacks charged residues. It
is predicted that this segment forms coil structures or
3.sub.1-helix structures. The mid-sized glycine-rich segment is
represented by the amino acid one letter consensus sequence
GQGGQGQG(G/R)Y GQG(A/S)G(S/G)S (SEQ ID NO: 6), and lacks charged
residues or contains one charged residue. It is predicted that this
segment forms coil structures. The shortest glycine-rich segment is
represented by the amino acid one letter consensus sequence
G(R/Q)GQG(G/R)YGQG (A/S/V)GGN (SEQ ID NO: 7). It is predicted that
this segment forms turn structures.
[0114] The repetitive fragment of the Euprosthenops australis MaSp1
protein is built up from alternating alanine-rich and glycine rich
segments, e.g.
TABLE-US-00004 (SEQ ID NO: 56) . . .
A.sub.1G.sub.1A.sub.2G.sub.2A.sub.3G.sub.3A.sub.4G.sub.4A.sub.5G.su-
b.5 . . .
It is observed that each of the above-identified shortest and
longest glycine-rich segments generally occur as every second
glycine-rich segment, e.g.
TABLE-US-00005 (SEQ ID NO: 56) . . . . . .
A.sub.1G.sub.shortA.sub.2G.sub.longA.sub.3G.sub.shortA.sub.4G.sub.l-
ongA.sub.5G.sub.short
In contrast, the less abundant, mid-sized glycine-rich fragment
generally occurs in between a glycine-rich segment of the longer
type and a glycine-rich segment of the shorter type, e.g.
TABLE-US-00006 (SEQ ID NO: 56) . . .
A.sub.1G.sub.shortA.sub.2G.sub.longA.sub.3G.sub.midA.sub.4G.sub.sho-
rtA.sub.5G.sub.long . . .
Example 3
Prediction of Secondary and Tertiary Structure of the Repetitive
Fragment of Euprosthenops australis Masp1 Protein
[0115] Spidroin polypeptides in solution typically fold by
formation of hairpin structures, which prefigures the anti-parallel
.beta.-sheet structure of the mature fiber. To discern possible
folding patterns for the repetitive fragment (SEQ ID NO: 3) of the
Euprosthenops australis MaSp1 protein of examples 1-2, protein
regions that are compatible with formation of hairpin or turn
structures were identified. The alanine-rich segments are unlikely
candidates for turn formation since they are predicted to form
helical structures, and more importantly, these segments are
generally held to make up the .beta.-sheets in the fiber.
[0116] Using a recently described algorithm for turn predictions
(Fuchs, P F & Alix, A J, High accuracy prediction of beta-turns
and their types using propensities and multiple alignments.
Proteins 59, 828-839 (2005)), the shortest glycine-rich segments
shows high likelihood for formation of type II .beta.-turns, while
the two longer glycine-rich segments are predicted to form coil
structures. The high content of GGX triplets in the longer Gly-rich
segments suggests that they can form 3.sub.1-helix structures.
[0117] The repetitive nature of the spidroin amino acid sequences
implies an equally repetitive nature of the folding pattern. Taken
together, these observations result in a folding of the repetitive
fragment of the Euprosthenops australis MaSp1 protein as shown in
FIG. 2A. It is notable that the positively charged residues almost
invariably are located in the proposed turn structures.
[0118] From the folding pattern of the repetitive fragment of the
Euprosthenops australis MaSp1 protein, a motif consisting of
alanine-rich segment/(longer) glycine-rich coil
segment/alanine-rich segment/(shorter) glycine-rich turn
segment/alanine-rich segment/(longer) glycine-rich coil
segment/alanine-rich segment, can be discerned (schematically
illustrated in FIG. 2A).
Example 4
Sequence Analysis of the Non-Repetitive C-Terminal Fragment of
MaSp1 Proteins
[0119] The primary structure of the C-terminal non-repetitive
fragment (SEQ ID NO: 4) of MaSp1 protein from Euprosthenops
australis, obtained in Example 1, was aligned with a number of
known C-terminal fragments of MaSp1 and MaSp2 proteins, inter alia
from Euprosthenops sp. (Pouchkina-Stantcheva, N N &
McQueen-Mason, S J, Molecular studies of a novel dragline silk from
a nursery web spider, Euprosthenops sp. (Pisauridae). Comp Biochem
Physiol B Biochem Mol Biol 138, 371-376 (2004)), Nephila clavipes
P19837-5 (Xu, M & Lewis, R V, Structure of a protein
superfiber: spider dragline silk. Proc Natl Acad Sci USA 87,
7120-7124 (1990)) and others.
[0120] From the alignment shown in FIG. 3, starting from the last
Ser in the repetitive fragment, it is evident that the C-terminal
regions of MaSp1 and MaSp2 are well conserved. Euprosthenops sp and
Nephila clavipes have 95% identical residues; Euprosthenops
australis and Nephila clavipes have 54% identical residues; and
Euprosthenops australis and Euprosthenops sp have 55% identical
residues. A consensus sequence of the C-terminal regions of MaSp1
and MaSp2 is provided as SEQ ID NO: 8. In FIG. 3, the following
MaSp proteins are aligned, denoted with GenBank accession entries
where applicable:
TABLE-US-00007 Species and spidroin protein Entry Euprosthenops sp
MaSp1 (Pouchkina- Cthyb_Esp Stantcheva, NN & McQueen-Mason, SJ,
ibid) Euprosthenops australis MaSp1 (SEQ ID NO: 4) CTnat_Eau
Argiope trifasciata MaSp1 AF350266_At1 Cyrtophora moluccensis Sp1
AY666062_Cm1 Latrodectus geometricus MaSp1 AF350273_Lg1 Latrodectus
hesperus MaSp1 AY953074_Lh1 Macrothele holsti Sp1 AY666068_Mh1
Nephila clavipes MaSp1 U20329_Nc1 Nephila pilipes MaSp1
AY666076_Np1 Nephila madagascariensis MaSp1 AF350277_Nm1 Nephila
senegalensis MaSp1 AF350279_Ns1 Octonoba varians Sp1 AY666057_Ov1
Psechrus sinensis Sp1 AY666064_Ps1 Tetragnatha kauaiensis MaSp1
AF350285_Tk1 Tetragnatha versicolor MaSp1 AF350286_Tv1 Araneus
bicentenarius Sp2 ABU20328_Ab2 Argiope amoena MaSp2 AY365016_Aam2
Argiope aurantia MaSp2 AF350263_Aau2 Argiope trifasciata MaSp2
AF350267_At2 Gasteracantha mammosa MaSp2 AF350272_Gm2 Latrodectus
geometricus MaSp2 AF350275_Lg2 Latrodectus hesperus MaSp2
AY953075_Lh2 Nephila clavipes MaSp2 AY654293_Nc2 Nephila
madagascariensis MaSp2 AF350278_Nm2 Nephila senegalensis MaSp2
AF350280_Ns2 Dolomedes tenebrosus Fb1 AF350269_DtFb1 Dolomedes
tenebrosus Fb2 AF350270_DtFb2 Araneus diadematus ADF-1 U47853_ADF1
Araneus diadematus ADF-2 U47854_ADF2 Araneus diadematus ADF-3
U47855_ADF3 Araneus diadematus ADF-4 U47856_ADF4
Example 5
Construction of MaSp1 Genes
[0121] A DNA sequence encoding the Euprosthenops australis-derived
protein 5Gly/Ala-CT.sub.nat (SEQ ID NO: 9) was amplified by PCR
with an Advantage GC2 kit (BD Biosciences, San Jose, Calif., USA),
using a MaSp1 clone from the cDNA library of Example 1 as template.
Restriction enzyme recognition sites BamHI and HindIII were
introduced at the 5'- and 3'-ends, respectively, and a stop codon
was introduced upstream of the HindIII site, by use of designed
primers. The BamHI-5Gly/Ala-CT.sub.nat-HindIII construct was then
subcloned into a modified pET32 vector (Merck Biosciences,
Darmstadt, Germany), prepared as described in Example 6(C)
below.
Example 6
Construction of Chimeric MaSp1 Genes (A) REP Gene Fragments
[0122] DNA sequences coding for partial repetitive fragments (REP)
denoted 3Gly/Ala and 4Gly/Ala were amplified by PCR with LA Taq
(TaKaRa Bio; Saint Germain-en-laye, France) in the presence of
betaine (Henke W et al, Betaine improves the PCR amplification of
GC-rich DNA sequences. Nucleic Acids Res 25, 3957-3958 (1997)),
using a partial cDNA clone encoding a repetitive region of
Euprosthenops sp MaSp1 protein (Pouchkina-Stantcheva, N N &
McQueen-Mason, S J, Molecular studies of a novel dragline silk from
a nursery web spider, Euprosthenops sp. (Pisauridae). Comp Biochem
Physiol B Biochem Mol Biol 138, 371-376 (2004)) (GenBank entry
CQ974358 or CQ816656) as template. Restriction enzyme recognition
sites were introduced at the 5'- and 3'-ends, giving the following
constructs: NcoI-3Gly/Ala-NheI and NcoI-4Gly/Ala-NheI to be joined
with a CT fragment (see below); and a NcoI-4Gly/Ala-XhoI clone to
be individually expressed, where a stop codon was inserted directly
upstream of the XhoI site.
(B) CT Gene Fragments
[0123] A DNA sequence coding for the non-repetitive C-terminal
domain from Euprosthenops sp (but with a high degree of similarity
to MaSp1 from Nephila clavipes and Nephila senegalensis) was
amplified by PCR using a genomic DNA clone encoding a C-terminal
MaSp1 domain (Pouchkina-Stantcheva, N N & McQueen-Mason, S J,
Molecular studies of a novel dragline silk from a nursery web
spider, Euprosthenops sp. (Pisauridae). Comp Biochem Physiol B
Biochem Mol Biol 138, 371-376 (2004)). Restriction enzyme
recognition sites were introduced at the 5'- and 3'-ends, giving
NheI-2Gly/Ala-CT.sub.hyb-XhoI, to be joined with the 3Gly/Ala and
4Gly/Ala partial REP clones (see above), and
NcoI-2Gly/Ala-CT.sub.hyb-XhoI, to be individually expressed.
(C) Construction of REP-CT Hybrid MaSp1 Genes
[0124] The 3Gly/Ala and 4Gly/Ala REP clones were joined with the CT
clones using the pCR.RTM. 2.1-TOPO.RTM. vector (Invitrogen). Then,
the resulting, fused 5Gly/Ala-CT.sub.hyb and 6Gly/Ala-CT.sub.hyb
clones were excised with NcoI and XhoI, and subcloned into a
modified pET32 vector (Novagen), where the original thrombin
cleavage site was removed and a new thrombin site was introduced
downstream of the enterokinase cleavage site.
Example 7
Expression of MaSp1 Fusion Proteins
[0125] The MaSp1 proteins coded for by the genes constructed in
examples 5-6 were expressed as fusion proteins (of the type
X-REP-CT) as follows, using a modified pET32 vector:
thioredoxin-tag/His-tag/S-tag/thrombin cleavage site/MaSp1 gene,
encoding a thioredoxin/His.sub.6/S-tag/thrombin cleavage site/MaSp1
protein, and an ampicillin resistance gene under control of the T7
promoter.
[0126] The different MaSp1 constructs in pET32 expression vectors
were transformed into Escherichia coli BL21(DE3) cells (Merck
Biosciences). The cells were grown at 30.degree. C. in
Luria-Bertani medium containing ampicillin to an OD.sub.600 of
1.0-1.5, induced with IPTG and further incubated for 4 h at room
temperature. The cells were harvested by centrifugation, and lysed
by DNAseI and lysozyme in 20 mM Tris-HCl, pH 8.0, 20 mM imidazole,
with 0.5 M NaCl, and further purified by His-tag affinity
chromatography on Ni-NTA agarose (Qiagen, West Sussex, UK). Bound
fusion proteins were eluted from the Ni-NTA column with 200 mM
imidazole in 20 mM Tris-HCl, pH 8.0, with 0.5 M NaCl, and dialyzed
against 20 mM Tris-HCl, pH 8.0. The resulting fusion proteins were
>90% pure as judged by coomassie-stained SDS polyacrylamide gels
and soluble in 20 mM Tris-HCl, pH 8.0. This process yielded
approximately 40 mg/l culture of fusion protein, which was stable
for weeks without significant precipitation.
[0127] In another experiment, the fusion proteins were expressed as
His.sub.6/thioredoxin/His.sub.6/thrombin cleavage site/MaSp1
proteins from a plasmid containing the corresponding gene and a
kanamycin resistance gene under control of the T7 promoter.
Example 8
Formation of Fibers from MaSp1 Proteins
[0128] Cleavage of the tags from the fusion proteins resulting from
Example 7, was performed in 20 mM Tris-HCl, pH 8, with a
thrombin:fusion protein ratio of 1:1000 (w/w), under very gentle
rocking at room temperature. Thrombin cleavage was complete within
30-60 min, as judged by SDS-PAGE. The resulting MaSp1 proteins
(FIG. 2B, SEQ ID NOS: 9-13) spontaneously polymerized into
macroscopic fibers to varying extents, see Table 1. The fibers were
initially formed at the water/air interface. The formation could be
observed by the naked eye from about 1 hour of incubation (see FIG.
4A, 4B), and after about 5 hours occurred no further fiber growth.
6Gly/Ala-CT.sub.hyb fibers were up to approximately 2 cm long, and
5Gly/Ala-CT.sub.nat fibers were .gtoreq.10 cm long. Repeated
experiments yielded 5Gly/Ala-CT.sub.nat fibers that were .gtoreq.20
cm (see FIG. 4C), and even .gtoreq.2 m long. Fiber formation could
be observed by the naked eye from about 10 min of incubation.
[0129] Fibers were isolated and washed with buffer and thereafter
subjected to N-terminal amino acid sequence analysis, which showed
only the sequence of the MaSp1 protein. This shows that the cleaved
tags are absent in the fibers.
Example 9
Analysis of MaSp1 Protein Fibers
A. Tensile Strength Measurements
[0130] The tensile strength of the 6Gly/Ala-CT.sub.hyb (SEQ ID NO:
13) and 5Gly/Ala-CT.sub.nat (SEQ ID NO: 9) fibers of example 8 was
determined as follows. In order to handle the shorter (1-2 cm)
6Gly/Ala-CT.sub.hyb fibers for tensile strength measurements, they
were incubated shortly in 15% glycerol in water before they were
air-dried. The longer (10 cm) 5Gly/Ala-CT.sub.nat fibers were
either untreated, incubated shortly in 15% glycerol, or drawn by
hand in 75% methanol before air-drying. Tensile strength of
air-dried fibers was measured by pulling the fibers in a Zwick
Material Tester at a rate of 10 mm/min. See Table 1.
[0131] The tensile strength of glycerol-treated air-dried 1-2 cm
long fibers from 6Gly/Ala-CT.sub.hyb (SEQ ID NO: 13) was about 2
MPa, and the strength of 10 cm fibers from 5Gly/Ala-CT.sub.nat (SEQ
ID NO: 9) was 4-5 MPa. Ten cm long 5Gly/Ala-CT.sub.nat fibers drawn
in the dehydrating solvent methanol before air-drying displayed a
tensile strength of 2-3 MPa, which is slightly less than for
glycerol-treated fibers of the same type. The highest tensile
strength now measured was 10 MPa, which was found for an air-dried
10 cm long 5Gly/Ala-CT.sub.nat fiber without further treatment.
[0132] The range of tensile strengths (2-10 MPa) is comparable to
the lower values reported for regenerated spider silk fibers (2-320
MPa). The longest spontaneously formed fibers derive from the
5Gly/Ala-CT.sub.nat construct, and such air-dried fibers also show
the greatest tensile strength. Potentially, this could be due to
its 12-15 residues long poly-Ala segments, relative the 8-14
residue Ala segments in 6Gly/Ala-CT.sub.hyb, which would give a
greater proportion of crystalline .beta.-sheet conformation in the
former protein.
TABLE-US-00008 TABLE 1 Fiber forming capacity of MaSp1 proteins SEQ
Fiber Fiber tensile ID Fiber forming length strength Protein NO
capacity (cm) (MPa) 5Gly/Ala-CT.sub.nat 9 ++++ .gtoreq.10 2-10
5Gly/Ala-CT.sub.nat 9 +++++ .gtoreq.20 180-230 (Example 11)
4Gly/Ala 10 aggregates n.a. n.a. 2Gly/Ala-CT.sub.hyb 11 aggregates
n.a. n.a. 5Gly/Ala-CT.sub.hyb 12 + .ltoreq.1 n.d.
6Gly/Ala-CT.sub.hyb 13 +++ 1-2 .ltoreq.2 n.a. = not applicable n.d.
= not determined
B. Scanning Electron Microscopy
[0133] The microscopic architecture of the 6Gly/Ala-CT.sub.hyb and
5Gly/Ala-CT.sub.nat fibers was analyzed with scanning electron
microscopy (SEM) (FIG. 5). Briefly, samples were applied on
SEM-stubs and vacuum-coated with a 6 nm layer of gold and
palladium. Specimens were observed and photographed in a LEO 1550
FEG SEM using an acceleration voltage of 10 kV.
[0134] This revealed diameters of 10-30 .mu.m for single fibers,
with individual fibers displaying rather homogenous diameters (FIG.
5a showing 6Gly/Ala-CT.sub.hyb, SEQ ID NO: 13). In addition to the
macroscopic fibers, gel-like particles were found. After air-drying
such particles of 6Gly/Ala-CT.sub.hyb directly on a SEM-stub,
fibers approximately 10-15 .mu.m in diameter were seen (FIG. 5b,
c). The diameter of macroscopic fibers of 5Gly/Ala-CT.sub.nat (SEQ
ID NO: 9), drawn in 75% methanol and air-dried, were 60-120 .mu.m
and they apparently contain several aligned fibers (FIG. 5d-f).
Fiber twisted before air-drying (FIG. 5e), end of fiber (FIG.
5f).
C. Circular Dichroism Spectroscopy
[0135] Fibers consisting of 6Gly/Ala-CT.sub.hyb protein (SEQ ID NO:
13) or 5Gly/Ala-CT.sub.nat (SEQ ID NO: 9), prepared in Example 8,
were washed in 20 mM phosphate buffer, pH 7, and suspended in 2%
SDS in the same buffer. Circular Dichroism spectra from 250 to 190
nm were recorded at 22.degree. C. in a 0.1 cm path length quartz
cuvette, using a Jasco J-810 spectropolarimeter. The scan speed was
50 nm/min, response time 2 sec, acquisition interval 0.1 nm, and
the band width 1 nm.
[0136] The spectrum shown in FIG. 6 is an accumulation of three
scans of fibers of 6Gly/Ala-CT.sub.hyb protein (SEQ ID NO: 13). It
displays a minimum at 220 nm and a maximum at 195 nm, features that
are characteristic of antiparallel 3-sheet structures. Highly
similar spectra were obtained for fibers of 5Gly/Ala-CT.sub.nat
(not shown). The spontaneously formed fibers thus exhibit similar
morphology and structure as native and regenerated spider silk
fibers.
Example 10
Biocompatibility of Recombinant Spider Silk
[0137] Since it is desirable to use spider silk fibers in
biomedical applications, the biocompatibility of the fibers has
been evaluated by an investigation of effects of recombinantly
produced silk using two different cell types.
[0138] The MaSp1 protein 5Gly/Ala-CT.sub.nat (SEQ ID NO: 9) was
expressed in bacteria as described in examples 7-8. Purified
protein was used to produce artificial silk fibers with lengths of
>10 cm, and even >20-200 cm, and diameters of around 100
.mu.m.
A. Embryonal Mouse Mast Cells
[0139] Embryonal (day 12.5) mouse mast cells (in vitro proliferated
for eight weeks using IL-8 and mast stem cell factor) were seeded
at two different cell densities, the higher density being about
four times the lower density. These cells do not adhere to the
plastic surface, but grow in suspension. Pieces of the silk fiber,
each about 0.5 cm long, were added to the wells. Mast cells were
incubated for three days, with or without the presence of silk
fiber, and thereafter living and dead cells were counted after
staining with Trypan blue (FIG. 7). The bars show the mean values
with standard error mean, n=2, each sample is counted in
triplicate.
[0140] The mast cells are not affected by the presence of the silk
fibers. After three days of growth, there are no significant
differences in cell death or proliferation compared to the negative
controls grown without silk fibers.
B. Human Embryonic Kidney (HEK) 293 Cells
[0141] Pieces of the silk fiber, about 0.5 cm long, were adsorbed
to the bottom of 6-well microtiter plates by letting them dry from
a small volume of buffer. The fibers do not detach when cell growth
media is added. Human embryonic kidney (HEK) 293 cells were then
plated at different cell densities and allowed to grow for a total
of six days. The HEK-293 cells adhere and grow attached to the
plastic cell surface. The ability of the HEK-293 cells to grow in
the proximity of the fibers, and the physical attachment of the
cells to the fibers was studied.
[0142] The HEK-293 cells attached and proliferated normally in the
wells containing silk fibers (as observed under the light
microscope). The cells grew very closely along the fiber edges, and
apparently even grew under a partly detached fiber (FIG. 8). After
seven days, the fibers were carefully detached from the plastic
surface, and it was clearly seen that groups of cells were
physically attached to the fibers. The fiber covers the upper right
half of the figure. HEK293-cells are seen attached to the edge of
the fiber, and also grow under the fiber.
[0143] The two different cell types (mast cells, HEK-293) studied
were not affected by the presence of recombinant silk fibers, even
at comparatively high amounts of silk. This indicates that the
tested artificial silk fibers resemble wild type dragline silk of
Euprosthenops australis, in being non-toxic and biocompatible. The
artificial silk fibers thus appear suitable for biomedical
applications.
Example 11
Mechanical Properties and Structure of MaSp1 Protein Fibers
[0144] The mechanical properties of fibers from 5Gly/Ala-CT.sub.nat
(SEQ ID NO: 9) were examined using tensile tests performed to yield
stress-strain curves (FIG. 9). The tensile properties were
characterized using a Zwick Roell Z2.5 material tester (Zwick, Ulm,
Germany). The tests were performed in air at ambient conditions
(20.degree. C. and 52% relative humidity) using a loading speed of
10 mm/min. Fiber pieces were transferred directly from buffer,
mounted and subjected to two stretching-relaxation cycles. In order
to generate a homogenous silk thread suitable for tensile testing,
the fibers were elongated using stretch-relaxation cycles. First,
the fibers were elongated by pulling up to a force of 0.1 N. After
relaxation, they were further drawn until a force of 0.25 N was
applied.
[0145] This treatment generated elongated homogenous fibers with a
diameter of approximately 80 .mu.m as determined by height
measurements using a Mitutoyo IDC-112B instrument (Mitutoyo Corp,
Tokyo, Japan) and confirmed by scanning electron microscopy (SEM)
as follows. Before and after stretch-relaxation cycles, fiber
pieces were applied on SEM stubs and air-dried overnight. The
samples were vacuum-coated with a 6 nm layer of gold and palladium.
Specimens were observed and photographed with a LEO 1550 FEG
microscope (Carl Zeiss, Oberkochen, Germany) using an acceleration
voltage of 10 kV.
[0146] The drawn fibers were cut into pieces, the ends of which
were fixed between cardboard paper with glue (Loctite 420, Loctite,
Goteborg, Sweden). Fiber samples were then fixed in the grips of
the material tester and stretched until they broke. Stress-strain
curves were constructed using the initial cross-sectional area of
the pre-drawn fibre, assuming a circular cross-section. The stress
values are normalized to the initial cross-sectional area of the
fiber. The strain corresponds to dL/L.sub.0 where L.sub.0 is the
initial length of the fiber and dL is the change in fiber length.
In FIG. 9, the stress-strain curves for three different samples of
double drawn fibers of 5Gly/Ala-CT.sub.nat (SEQ ID NO: 9) are
shown, and their tensile strength measured approximately 0.2
GPa.
[0147] The microscopic architecture of the fibers was analyzed by
SEM (FIG. 10). The spontaneously formed fibers have a homogenous
flattened appearance and a width of up to several hundred
micrometers, while the height measures some ten micrometers (FIG.
10a,b).
[0148] After the fibers had been subjected to stretch-relaxation
cycles, their cross section adopted a more rounded shape with a
compact substructure of tightly aligned fibrils (FIG. 10c-f). The
appearance of cut or fractured surfaces (FIG. 10e,f) further attest
to the compactness of the produced fiber.
[0149] In conclusion, the spontaneously formed fibers show similar
morphology and mechanical properties as native or regenerated
spider silk fibers, even without spinning.
Example 12
Spidroin Protein Variants
[0150] Strong intermolecular interactions are thought to contribute
to the impressive tensile strength of spider silk. Therefore,
variants of miniature spidroins that allow intermolecular covalent
cross-linking in the fibers have been produced. Two different
mutant spidroin proteins have been constructed by site-directed
mutagenesis to introduce two cysteine residues in the first (SEQ ID
NO: 14, positions 36 and 37) and the fourth (SEQ ID NO: 15,
positions 128 and 129) alanine block, respectively. These variants
have been expressed and isolated using the same protocol as
described in Examples 7-8 for the genes constructed in Examples
5-6.
These variants (SEQ ID NOS: 14-15) form fibers in the same manner
as 5Gly/Ala-CT.sub.nat (SEQ ID NO: 9).
[0151] In order to elucidate the importance of dimerization of the
C-terminal domain, a variant where the cysteine residue in the
C-terminal domain is exchanged for a serine residue has been
constructed (SEQ ID NO: 16, position 222). However, this variant
(SEQ ID NO: 16) forms fibers in the same manner as
5Gly/Ala-CT.sub.nat (SEQ ID NO: 9).
Example 13
Removal of LPS and Other Pyrogens from
[0152] Expressed Spidroin Proteins E. coli cells expressing the
desired spidroin fusion protein are washed with the following
buffers:
A: 100 mM Tris, pH 8,
B: 5 mM CaCl.sub.2, 100 mM Tris, pH 8,
C: 10 mM EDTA, 100 mM Tris, pH 8,
D: 100 mM Tris, pH 8, and
E: 100 mM Tris, pH 8.
[0153] Thereafter, the cells are lysed in 20 mM Tris, pH 8
supplemented with lysozyme and DNaseI. The protein sample is then
loaded on a Ni-sepharose matrix and washed with 20 mM Tris, 10-100
mM imidazole, pH 8 before elution with 20 mM Tris, 100-300 mM
imidazole, pH 8. Relevant fractions are pooled and dialyzed against
20 mM Tris, pH 8 over night. The protein sample is then
supplemented with 100 .mu.M CaCl.sub.2 and finally passed through
an EndoTrap Blue column, previously equilibrated with 20 mM Tris,
100 .mu.M CaCl.sub.2, pH 8. In this way, protein samples with a
pyrogen content of 1 EU/mg protein can be obtained, as judged by
IPT and a LAL kinetic assay.
[0154] The fusion protein is then proteolytically cleaved with
thrombin using a 1:1000 (w/w) thrombin:fusion protein ratio, which
induces fiber formation (as described above). The fibers are washed
3 times in 20 mM Tris, pH 8 and finally 3 times in water. This
gives fibers with a pyrogen content of 0.25 EU/mg fiber.
[0155] The structural characteristics of the fibers are unaffected
after autoclaving at 125.degree. C. and 1.5 bar for 10 min, which
enables efficient sterilization of the material. The fibers are
chemically stable and can not be solubilized in either of 8 M urea,
6 M GuaHCl, or neat HAc. However, the fibers can be solubilized in
neat HFIP or formic acid.
Sequence CWU 1
1
7213766DNAEuprosthenops australis 1gtcaaggtgc tggaggtaat gccgctgcag
cagccgcagc agcagcagca gcagcagctg 60gacagggcgg tcaaggtgga tatggtggac
taggtcaagg aggatatgga cagggtgcag 120gaagttctgc agccgccgcc
gccgcagcag cagcagcagc tgcagcagct ggacgaggtc 180aaggaggata
tggtcaaggt tctggaggta atgccgctgc agcagccgca gcagctgcag
240cagcagcatc tggacaagga ggtcaaggag gacaaggtgg acaaggtcaa
ggtggatatg 300gacaaggtgc aggaagttct gcagccgccg ccgccgcagc
agcagcagcc gccgcagcag 360ctggacaagg tcaaggacga tatggtcaag
gtgctggagg taatgccgct gcagcagccg 420cagcagctgc agcagcagca
gctggacaag gaggtcaagg aggacaaggt ggactaggtc 480aaggaggata
tggacaaggt gcaggaagtt ctgcagccgc cgccgcagca tcagcagccg
540ccgcagcagc tggacgaggt caaggaggat atggtcaagg tgctggaggt
aatgccgctg 600cagcagccgc agcagctgcc gccgccgcag cagctggaca
gggtggtcaa ggtggatatg 660gtggactagg tcaaggagga tatggacaag
gtgcaggaag ttctgcagcc gctgccgccg 720cagcagcagc agccgccgcc
gcaggtggac aaggtggaca aggtcaagga agatatggac 780aaggtgcagg
aagttctgca gccgctgccg ccgcagcagc agcagccgcc gcagcagctg
840gacaaggtca aggaggatat ggtcaaggtg ctggaggtaa tgccgctgca
gcagccgcag 900cagctgcagc agcagcagct ggacaaggag gtcaaggagg
acaaggtgga ctaggtcaag 960gaggatatgg acaaggtgca ggaagttctg
ccgccgccgc cgcagcagca gcagccgccg 1020cagcagctgg acgaggtcaa
ggaggatatg gtcaaggtgc tggaggtaat gccgctgcag 1080cagccgcagc
agctgccgaa gccgcagcag ctggacaggg tggtcaaggt ggatatggtg
1140gactaggtca aggaggatat ggacaaggtg caggaagttc tgcagccgcc
gccgcagcag 1200cagcagccgc cgcagcagct ggacgaggtc aaggaggata
tggtcaaggt gctggaggta 1260atgccgctgc agcagccgca gcagctgccg
ccgccgcagc agctggacag ggtggtcaag 1320gtggatatgg tggactaggt
caaggaggat atggacaagg tgcaggaagt tctgcagccg 1380ctgccgccgc
agcagcagca gccgccgccg caggtggaca aggtggacaa ggtcaaggaa
1440gatatggaca aggtgcagga agttctgcag ccgctgccgc cgcagcagca
gcagcagccg 1500cagcagctgg acgaggtcaa ggaggatatg gtcaaggttc
tggaggtaat gccgctgcag 1560cagccgcagc agctgcagca gcagcatctg
gacaaggaag tcaaggagga caaggtggac 1620aaggtcaagg tggatatgga
caaggtgcag gaagttctgc agccgccgcc gccgccgcag 1680cagcagccgc
cgcagcatct ggacgaggtc aaggaggata tggtcaaggt gctggaggta
1740atgccgctgc tgcagccgca gcagctgccg ccgccgcagc agctggacag
ggcggtcaag 1800gtggatatgg tggactaggt caaggaggat atggacaagg
tgcaggaagt tctgcagccg 1860ctgccgccgc cgcagcagcc gccgcagcag
gtggacaagg tggacaaggt caaggaggat 1920atggacaagg tgcaggaagt
tctgcagccg ccgccgcagc agcagcagca gcagccgcag 1980cagctggacg
aggtcaagga ggatatggtc aaggttctgg aggtaatgcc gctgcagcag
2040ccgcagcagc tgcagcagca gcatctggac aaggaggtca aggaggacaa
ggtggacaag 2100gtcaaggtgg rtatggacaa ggtgcaggaa gttctgcagc
cgccgccgcc gcagcagcag 2160cagccgccgc agcagctgga caaggtcaag
gaggatatgg tcaaggtgct ggaggtaatg 2220ccgctgcagc agccgcagca
gctgcagcag cagcagctgg acaaggaggt caaggaggac 2280aaggtggact
aggtcaagga ggatatggac aaggtgcagg aagttctgca gccgccgccg
2340cagcmgcmgc agcagccgcc gcagcagctg gacgaggtca aggaggatat
ggtcaaggtg 2400ttggaggtaa tgccgctgca gcagccgcag cagctgcagc
agcagcagct ggacaaggag 2460gtcaaggagg acaaggtgga ctaggtcaag
gaggatatgg acaaggtgca ggtagttctg 2520cagccgccgc cgccgccgca
gcagcagccg ccgcagcagc tggacgaggt caaggaggat 2580atggtcaagg
ttctggaggt aatgccgctg cagcagccgc agcagctgca gcagcagcat
2640ctggacaagg aagtcaagga ggacaaggtg gacaaggtca aggtggatat
ggacaaggtg 2700caggaagttc tgcagccgcc gccgccgcag cagcagcagc
cgccgcagca tctggacgag 2760gtcaaggagg atatggtcaa ggtgctggag
gtaatgccgc tgctgcagcc gcagcagctg 2820ccgccgccgc agcagctgga
cagggcggtc aaggtggata tggtggacta ggtcaaggag 2880gatatggaca
aggtgcagga agttctgcag ccgctgccgc cgccgcagca gccgccgcag
2940caggtggaca aggtggacaa ggtcaaggag gatatggaca aggttcagga
ggttctgcag 3000ccgccgccgc cgccgcagca gcagcagcag ctgcagcagc
tggacgaggt caaggaggat 3060atggtcaagg ttctggaggt aatgctgctg
ccgcagccgc tgccgccgcc gccgccgctg 3120cagcagccgg acagggaggt
caaggtggat atggtagaca aagccaaggt gctggttccg 3180ctgctgctgc
tgctgctgct gctgccgctg ctgctgctgc aggatctgga caaggtggat
3240acggtggaca aggtcaagga ggttatggtc agagtagtgc ttctgcttca
gctgctgcgt 3300cagctgctag tactgtagct aattcggtga gtcgcctctc
atcgccttcc gcagtatctc 3360gagtttcttc agcagtttct agcttggttt
caaatggtca agtgaatatg gcagcgttac 3420ctaatatcat ttccaacatt
tcttcttctg tcagtgcatc tgctcctggt gcttctggat 3480gtgaggtcat
agtgcaagct ctactcgaag tcatcactgc tcttgttcaa atcgttagtt
3540cttctagtgt tggatatatt aatccatctg ctgtgaacca aattactaat
gttgttgcta 3600atgccatggc tcaagtaatg ggctgaggtt tttaatagta
aaaggtgtga tattcctcaa 3660tgttttgaaa attattaatc gaatttttac
cttgtgtgct atcagatata aattgaagta 3720taataaataa atatttgcat
tttcaaaaaa aaaaaaaaaa aaaaaa 376621207PRTEuprosthenops australis
2Gln Gly Ala Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala1 5
10 15Ala Ala Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gly Leu Gly
Gln 20 25 30Gly Gly Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala
Ala Ala 35 40 45Ala Ala Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly
Gly Tyr Gly 50 55 60Gln Gly Ser Gly Gly Asn Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala65 70 75 80Ala Ala Ser Gly Gln Gly Gly Gln Gly Gly
Gln Gly Gly Gln Gly Gln 85 90 95Gly Gly Tyr Gly Gln Gly Ala Gly Ser
Ser Ala Ala Ala Ala Ala Ala 100 105 110Ala Ala Ala Ala Ala Ala Ala
Ala Gly Gln Gly Gln Gly Arg Tyr Gly 115 120 125Gln Gly Ala Gly Gly
Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 130 135 140Ala Ala Ala
Gly Gln Gly Gly Gln Gly Gly Gln Gly Gly Leu Gly Gln145 150 155
160Gly Gly Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala
165 170 175Ser Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr
Gly Gln 180 185 190Gly Ala Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala 195 200 205Ala Ala Ala Gly Gln Gly Gly Gln Gly Gly
Tyr Gly Gly Leu Gly Gln 210 215 220Gly Gly Tyr Gly Gln Gly Ala Gly
Ser Ser Ala Ala Ala Ala Ala Ala225 230 235 240Ala Ala Ala Ala Ala
Ala Ala Gly Gly Gln Gly Gly Gln Gly Gln Gly 245 250 255Arg Tyr Gly
Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala 260 265 270Ala
Ala Ala Ala Ala Ala Ala Gly Gln Gly Gln Gly Gly Tyr Gly Gln 275 280
285Gly Ala Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
290 295 300Ala Ala Gly Gln Gly Gly Gln Gly Gly Gln Gly Gly Leu Gly
Gln Gly305 310 315 320Gly Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala
Ala Ala Ala Ala Ala 325 330 335Ala Ala Ala Ala Ala Ala Gly Arg Gly
Gln Gly Gly Tyr Gly Gln Gly 340 345 350Ala Gly Gly Asn Ala Ala Ala
Ala Ala Ala Ala Ala Ala Glu Ala Ala 355 360 365Ala Ala Gly Gln Gly
Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gln Gly 370 375 380Gly Tyr Gly
Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala385 390 395
400Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly Gln Gly
405 410 415Ala Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala 420 425 430Ala Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gly
Leu Gly Gln Gly 435 440 445Gly Tyr Gly Gln Gly Ala Gly Ser Ser Ala
Ala Ala Ala Ala Ala Ala 450 455 460Ala Ala Ala Ala Ala Ala Gly Gly
Gln Gly Gly Gln Gly Gln Gly Arg465 470 475 480Tyr Gly Gln Gly Ala
Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala 485 490 495Ala Ala Ala
Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly Gln Gly 500 505 510Ser
Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 515 520
525Ser Gly Gln Gly Ser Gln Gly Gly Gln Gly Gly Gln Gly Gln Gly Gly
530 535 540Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala
Ala Ala545 550 555 560Ala Ala Ala Ala Ala Ser Gly Arg Gly Gln Gly
Gly Tyr Gly Gln Gly 565 570 575Ala Gly Gly Asn Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala 580 585 590Ala Ala Gly Gln Gly Gly Gln
Gly Gly Tyr Gly Gly Leu Gly Gln Gly 595 600 605Gly Tyr Gly Gln Gly
Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala 610 615 620Ala Ala Ala
Ala Ala Gly Gly Gln Gly Gly Gln Gly Gln Gly Gly Tyr625 630 635
640Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala
645 650 655Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly Gln
Gly Ser 660 665 670Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ser 675 680 685Gly Gln Gly Gly Gln Gly Gly Gln Gly Gly
Gln Gly Gln Gly Gly Tyr 690 695 700Gly Gln Gly Ala Gly Ser Ser Ala
Ala Ala Ala Ala Ala Ala Ala Ala705 710 715 720Ala Ala Ala Ala Ala
Gly Gln Gly Gln Gly Gly Tyr Gly Gln Gly Ala 725 730 735Gly Gly Asn
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 740 745 750Gly
Gln Gly Gly Gln Gly Gly Gln Gly Gly Leu Gly Gln Gly Gly Tyr 755 760
765Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala
770 775 780Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly Gln
Gly Val785 790 795 800Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala 805 810 815Gly Gln Gly Gly Gln Gly Gly Gln Gly
Gly Leu Gly Gln Gly Gly Tyr 820 825 830Gly Gln Gly Ala Gly Ser Ser
Ala Ala Ala Ala Ala Ala Ala Ala Ala 835 840 845Ala Ala Ala Ala Ala
Gly Arg Gly Gln Gly Gly Tyr Gly Gln Gly Ser 850 855 860Gly Gly Asn
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ser865 870 875
880Gly Gln Gly Ser Gln Gly Gly Gln Gly Gly Gln Gly Gln Gly Gly Tyr
885 890 895Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala
Ala Ala 900 905 910Ala Ala Ala Ala Ser Gly Arg Gly Gln Gly Gly Tyr
Gly Gln Gly Ala 915 920 925Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala 930 935 940Ala Gly Gln Gly Gly Gln Gly Gly
Tyr Gly Gly Leu Gly Gln Gly Gly945 950 955 960Tyr Gly Gln Gly Ala
Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala 965 970 975Ala Ala Ala
Ala Gly Gly Gln Gly Gly Gln Gly Gln Gly Gly Tyr Gly 980 985 990Gln
Gly Ser Gly Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 995
1000 1005Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly Gln
Gly 1010 1015 1020Ser Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala 1025 1030 1035Ala Ala Ala Ala Gly Gln Gly Gly Gln Gly
Gly Tyr Gly Arg Gln 1040 1045 1050Ser Gln Gly Ala Gly Ser Ala Ala
Ala Ala Ala Ala Ala Ala Ala 1055 1060 1065Ala Ala Ala Ala Ala Gly
Ser Gly Gln Gly Gly Tyr Gly Gly Gln 1070 1075 1080Gly Gln Gly Gly
Tyr Gly Gln Ser Ser Ala Ser Ala Ser Ala Ala 1085 1090 1095Ala Ser
Ala Ala Ser Thr Val Ala Asn Ser Val Ser Arg Leu Ser 1100 1105
1110Ser Pro Ser Ala Val Ser Arg Val Ser Ser Ala Val Ser Ser Leu
1115 1120 1125Val Ser Asn Gly Gln Val Asn Met Ala Ala Leu Pro Asn
Ile Ile 1130 1135 1140Ser Asn Ile Ser Ser Ser Val Ser Ala Ser Ala
Pro Gly Ala Ser 1145 1150 1155Gly Cys Glu Val Ile Val Gln Ala Leu
Leu Glu Val Ile Thr Ala 1160 1165 1170Leu Val Gln Ile Val Ser Ser
Ser Ser Val Gly Tyr Ile Asn Pro 1175 1180 1185Ser Ala Val Asn Gln
Ile Thr Asn Val Val Ala Asn Ala Met Ala 1190 1195 1200Gln Val Met
Gly 120531110PRTEuprosthenops
australisREPEAT(7)..(19)REPEAT(20)..(42)REPEAT(43)..(56)REPEAT(57)..(70)R-
EPEAT(71)..(83)REPEAT(84)..(106)REPEAT(107)..(120)REPEAT(121)..(134)REPEAT-
(135)..(147)REPEAT(148)..(170)REPEAT(171)..(183)REPEAT(184)..(197)REPEAT(1-
98)..(211)REPEAT(212)..(234)REPEAT(235)..(248)REPEAT(249)..(265)REPEAT(266-
)..(279)REPEAT(280)..(293)REPEAT(294)..(306)REPEAT(307)..(329)REPEAT(330).-
.(342)REPEAT(343)..(356)REPEAT(357)..(370)REPEAT(371)..(393)REPEAT(394)..(-
406)REPEAT(407)..(420)REPEAT(421)..(434)REPEAT(435)..(457)REPEAT(458)..(47-
0)REPEAT(471)..(488)REPEAT(489)..(502)REPEAT(503)..(516)REPEAT(517)..(529)-
REPEAT(530)..(552)REPEAT(553)..(566)REPEAT(567)..(580)REPEAT(581)..(594)RE-
PEAT(595)..(617)REPEAT(618)..(630)REPEAT(631)..(647)REPEAT(648)..(661)REPE-
AT(662)..(675)REPEAT(676)..(688)REPEAT(689)..(711)REPEAT(712)..(725)REPEAT-
(726)..(739)REPEAT(740)..(752)REPEAT(753)..(775)REPEAT(776)..(789)REPEAT(7-
90)..(803)REPEAT(804)..(816)REPEAT(817)..(839)REPEAT(840)..(853)REPEAT(854-
)..(867)REPEAT(868)..(880)REPEAT(881)..(903)REPEAT(904)..(917)REPEAT(918).-
.(931)REPEAT(932)..(945)REPEAT(946)..(968)REPEAT(969)..(981)REPEAT(982)..(-
998)REPEAT(999)..(1013)REPEAT(1014)..(1027)REPEAT(1028)..(1042)REPEAT(1043-
)..(1059)REPEAT(1060)..(1073)REPEAT(1074)..(1092) 3Gln Gly Ala Gly
Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala1 5 10 15Ala Ala Ala
Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gln 20 25 30Gly Gly
Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala 35 40 45Ala
Ala Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly 50 55
60Gln Gly Ser Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala65
70 75 80Ala Ala Ser Gly Gln Gly Gly Gln Gly Gly Gln Gly Gly Gln Gly
Gln 85 90 95Gly Gly Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala
Ala Ala 100 105 110Ala Ala Ala Ala Ala Ala Ala Ala Gly Gln Gly Gln
Gly Arg Tyr Gly 115 120 125Gln Gly Ala Gly Gly Asn Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala 130 135 140Ala Ala Ala Gly Gln Gly Gly Gln
Gly Gly Gln Gly Gly Leu Gly Gln145 150 155 160Gly Gly Tyr Gly Gln
Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala 165 170 175Ser Ala Ala
Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly Gln 180 185 190Gly
Ala Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 195 200
205Ala Ala Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gln
210 215 220Gly Gly Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala
Ala Ala225 230 235 240Ala Ala Ala Ala Ala Ala Ala Gly Gly Gln Gly
Gly Gln Gly Gln Gly 245 250 255Arg Tyr Gly Gln Gly Ala Gly Ser Ser
Ala Ala Ala Ala Ala Ala Ala 260 265 270Ala Ala Ala Ala Ala Ala Ala
Gly Gln Gly Gln Gly Gly Tyr Gly Gln 275 280 285Gly Ala Gly Gly Asn
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 290 295 300Ala Ala Gly
Gln Gly Gly Gln Gly Gly Gln Gly Gly Leu Gly Gln Gly305 310 315
320Gly Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala
325 330 335Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly
Gln Gly 340 345 350Ala Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala
Ala Glu Ala Ala 355 360 365Ala Ala Gly Gln Gly Gly Gln Gly Gly Tyr
Gly Gly Leu Gly Gln Gly 370 375 380Gly Tyr Gly Gln Gly Ala Gly Ser
Ser Ala Ala Ala Ala Ala Ala Ala385 390 395 400Ala Ala Ala Ala Ala
Ala Gly Arg Gly Gln Gly Gly Tyr Gly Gln Gly 405 410 415Ala Gly Gly
Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 420 425 430Ala
Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gln Gly 435 440
445Gly Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala
450 455 460Ala Ala Ala Ala Ala Ala Gly Gly
Gln Gly Gly Gln Gly Gln Gly Arg465 470 475 480Tyr Gly Gln Gly Ala
Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala 485 490 495Ala Ala Ala
Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly Gln Gly 500 505 510Ser
Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 515 520
525Ser Gly Gln Gly Ser Gln Gly Gly Gln Gly Gly Gln Gly Gln Gly Gly
530 535 540Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala
Ala Ala545 550 555 560Ala Ala Ala Ala Ala Ser Gly Arg Gly Gln Gly
Gly Tyr Gly Gln Gly 565 570 575Ala Gly Gly Asn Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala 580 585 590Ala Ala Gly Gln Gly Gly Gln
Gly Gly Tyr Gly Gly Leu Gly Gln Gly 595 600 605Gly Tyr Gly Gln Gly
Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala 610 615 620Ala Ala Ala
Ala Ala Gly Gly Gln Gly Gly Gln Gly Gln Gly Gly Tyr625 630 635
640Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala
645 650 655Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly Gln
Gly Ser 660 665 670Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ser 675 680 685Gly Gln Gly Gly Gln Gly Gly Gln Gly Gly
Gln Gly Gln Gly Gly Tyr 690 695 700Gly Gln Gly Ala Gly Ser Ser Ala
Ala Ala Ala Ala Ala Ala Ala Ala705 710 715 720Ala Ala Ala Ala Ala
Gly Gln Gly Gln Gly Gly Tyr Gly Gln Gly Ala 725 730 735Gly Gly Asn
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 740 745 750Gly
Gln Gly Gly Gln Gly Gly Gln Gly Gly Leu Gly Gln Gly Gly Tyr 755 760
765Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala
770 775 780Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly Gln
Gly Val785 790 795 800Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala 805 810 815Gly Gln Gly Gly Gln Gly Gly Gln Gly
Gly Leu Gly Gln Gly Gly Tyr 820 825 830Gly Gln Gly Ala Gly Ser Ser
Ala Ala Ala Ala Ala Ala Ala Ala Ala 835 840 845Ala Ala Ala Ala Ala
Gly Arg Gly Gln Gly Gly Tyr Gly Gln Gly Ser 850 855 860Gly Gly Asn
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ser865 870 875
880Gly Gln Gly Ser Gln Gly Gly Gln Gly Gly Gln Gly Gln Gly Gly Tyr
885 890 895Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala
Ala Ala 900 905 910Ala Ala Ala Ala Ser Gly Arg Gly Gln Gly Gly Tyr
Gly Gln Gly Ala 915 920 925Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala 930 935 940Ala Gly Gln Gly Gly Gln Gly Gly
Tyr Gly Gly Leu Gly Gln Gly Gly945 950 955 960Tyr Gly Gln Gly Ala
Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala 965 970 975Ala Ala Ala
Ala Gly Gly Gln Gly Gly Gln Gly Gln Gly Gly Tyr Gly 980 985 990Gln
Gly Ser Gly Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 995
1000 1005Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly Gln
Gly 1010 1015 1020Ser Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala 1025 1030 1035Ala Ala Ala Ala Gly Gln Gly Gly Gln Gly
Gly Tyr Gly Arg Gln 1040 1045 1050Ser Gln Gly Ala Gly Ser Ala Ala
Ala Ala Ala Ala Ala Ala Ala 1055 1060 1065Ala Ala Ala Ala Ala Gly
Ser Gly Gln Gly Gly Tyr Gly Gly Gln 1070 1075 1080Gly Gln Gly Gly
Tyr Gly Gln Ser Ser Ala Ser Ala Ser Ala Ala 1085 1090 1095Ala Ser
Ala Ala Ser Thr Val Ala Asn Ser Val Ser 1100 1105
1110497PRTEuprosthenops australis 4Arg Leu Ser Ser Pro Ser Ala Val
Ser Arg Val Ser Ser Ala Val Ser1 5 10 15Ser Leu Val Ser Asn Gly Gln
Val Asn Met Ala Ala Leu Pro Asn Ile 20 25 30Ile Ser Asn Ile Ser Ser
Ser Val Ser Ala Ser Ala Pro Gly Ala Ser 35 40 45Gly Cys Glu Val Ile
Val Gln Ala Leu Leu Glu Val Ile Thr Ala Leu 50 55 60Val Gln Ile Val
Ser Ser Ser Ser Val Gly Tyr Ile Asn Pro Ser Ala65 70 75 80Val Asn
Gln Ile Thr Asn Val Val Ala Asn Ala Met Ala Gln Val Met 85 90
95Gly523PRTArtificial SequenceConsensus sequence derived from
internal repeats of Euprosthenops australis MaSp1 5Gly Gln Gly Gly
Gln Gly Gly Gln Gly Gly Leu Gly Gln Gly Gly Tyr1 5 10 15Gly Gln Gly
Ala Gly Ser Ser 20617PRTArtificial SequenceConsensus sequence
derived from internal repeats of Euprosthenops australis MaSp1 6Gly
Gln Gly Gly Gln Gly Gln Gly Gly Tyr Gly Gln Gly Ala Gly Ser1 5 10
15Ser714PRTArtificial SequenceConsensus sequence derived from
internal repeats of Euprosthenops australis MaSp1 7Gly Arg Gly Gln
Gly Gly Tyr Gly Gln Gly Ala Gly Gly Asn1 5 108100PRTArtificial
SequenceConsensus sequence derived from known MaSp1 and MaSp2
proteins 8Ser Arg Leu Ser Ser Pro Gln Ala Ser Ser Arg Val Ser Ser
Ala Val1 5 10 15Ser Asn Leu Val Ser Ser Gly Pro Thr Asn Ser Ala Ala
Leu Ser Asn 20 25 30Thr Ile Ser Asn Val Val Ser Gln Ile Ser Ala Ser
Asn Pro Gly Leu 35 40 45Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu
Glu Val Val Ser Ala 50 55 60Leu Val His Ile Leu Gly Ser Ser Ser Ile
Gly Gln Val Asn Tyr Gly65 70 75 80Ser Ala Gly Gln Ala Thr Gln Ile
Val Gly Gln Ser Val Ala Gln Ala 85 90 95Leu Gly Glu Phe
1009269PRTArtificial SequenceSequence derived from Euprosthenops
australis MaSp1 9Gly Ser Ala Met Gly Tyr Leu Trp Ile Gln Gly Gln
Gly Gly Tyr Gly1 5 10 15Gly Leu Gly Gln Gly Gly Tyr Gly Gln Gly Ala
Gly Ser Ser Ala Ala 20 25 30Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Gly Gly Gln Gly Gly Gln 35 40 45Gly Gln Gly Gly Tyr Gly Gln Gly Ser
Gly Gly Ser Ala Ala Ala Ala 50 55 60Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Gly Arg Gly Gln Gly65 70 75 80Gly Tyr Gly Gln Gly Ser
Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala 85 90 95Ala Ala Ala Ala Ala
Ala Ala Ala Gly Gln Gly Gly Gln Gly Gly Tyr 100 105 110Gly Arg Gln
Ser Gln Gly Ala Gly Ser Ala Ala Ala Ala Ala Ala Ala 115 120 125Ala
Ala Ala Ala Ala Ala Ala Gly Ser Gly Gln Gly Gly Tyr Gly Gly 130 135
140Gln Gly Gln Gly Gly Tyr Gly Gln Ser Ser Ala Ser Ala Ser Ala
Ala145 150 155 160Ala Ser Ala Ala Ser Thr Val Ala Asn Ser Val Ser
Arg Leu Ser Ser 165 170 175Pro Ser Ala Val Ser Arg Val Ser Ser Ala
Val Ser Ser Leu Val Ser 180 185 190Asn Gly Gln Val Asn Met Ala Ala
Leu Pro Asn Ile Ile Ser Asn Ile 195 200 205Ser Ser Ser Val Ser Ala
Ser Ala Pro Gly Ala Ser Gly Cys Glu Val 210 215 220Ile Val Gln Ala
Leu Leu Glu Val Ile Thr Ala Leu Val Gln Ile Val225 230 235 240Ser
Ser Ser Ser Val Gly Tyr Ile Asn Pro Ser Ala Val Asn Gln Ile 245 250
255Thr Asn Val Val Ala Asn Ala Met Ala Gln Val Met Gly 260
26510126PRTEuprosthenops australis 10Gly Ser Ala Met Gly Gln Gly
Ser Gly Gly Asn Ala Ala Ala Ala Ala1 5 10 15Ala Ala Ala Ala Ala Ala
Ala Ala Ser Gly Gln Gly Gly Gln Gly Gly 20 25 30Gln Gly Gln Gly Gly
Tyr Gly Gln Gly Ala Gly Ile Ser Ala Ala Ala 35 40 45Ala Ala Ala Ala
Ala Ala Ala Ala Gly Ala Ala Ala Gly Arg Gly Gln 50 55 60Gly Gly Tyr
Gly Gln Gly Ala Gly Gly Asn Ala Ala Ala Ala Ala Ala65 70 75 80Ala
Ala Ala Ala Ala Ala Ala Ala Gly Gln Gly Gly Gln Gly Gly Tyr 85 90
95Gly Gly Gln Gly Leu Gly Gly Tyr Gly Gln Gly Ala Gly Ser Ser Ala
100 105 110Ala Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln Ser Val Tyr
115 120 12511152PRTArtificial SequenceHybrid sequence derived from
Euprosthenops australis and Euprosthenops sp MaSp1 11Gly Ser Ala
Met Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly1 5 10 15Gly Gln
Gly Ala Gly Arg Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala 20 25 30Ala
Ala Gly Gly Ser Gly Gln Gly Gly Tyr Gly Gly Val Gly Ser Gly 35 40
45Ala Ser Ala Ala Ser Ala Ala Ala Ser Arg Leu Ser Ser Pro Glu Ala
50 55 60Ser Ser Arg Val Ser Ser Ala Val Ser Asn Leu Val Ser Ser Gly
Pro65 70 75 80Thr Asn Ser Ala Ala Leu Ser Ser Thr Ile Ser Asn Val
Val Ser Gln 85 90 95Ile Gly Ala Ser Asn Pro Gly Leu Ser Gly Cys Asp
Val Leu Val Gln 100 105 110Ala Leu Leu Glu Val Val Ser Ala Leu Ile
His Ile Leu Gly Ser Ser 115 120 125Ser Ile Gly Gln Val Asn Tyr Gly
Ser Ala Gly Gln Ala Thr Gln Leu 130 135 140Val Gly Gln Ser Val Tyr
Gln Ala145 15012257PRTArtificial SequenceHybrid sequence derived
from Euprosthenops australis and Euprosthenops sp MaSp1 12Gly Ser
Ala Met Ala Ser Gly Gln Gly Gly Gln Gly Gly Gln Gly Gln1 5 10 15Gly
Gly Tyr Gly Gln Gly Ala Gly Ile Ser Ala Ala Ala Ala Ala Ala 20 25
30Ala Ala Ala Ala Ala Gly Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr
35 40 45Gly Gln Gly Ala Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala
Ala 50 55 60Ala Ala Ala Ala Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly
Gly Gln65 70 75 80Gly Leu Gly Gly Tyr Gly Gln Gly Ala Gly Ser Ser
Ala Ala Ala Ala 85 90 95Ala Ala Ala Ala Gly Arg Gly Gln Ser Val Tyr
Ala Ser Gly Gly Ala 100 105 110Gly Gln Gly Gly Tyr Gly Gly Leu Gly
Gly Gln Gly Ala Gly Arg Gly 115 120 125Gly Gln Gly Ala Gly Ala Ala
Ala Ala Ala Ala Gly Gly Ser Gly Gln 130 135 140Gly Gly Tyr Gly Gly
Val Gly Ser Gly Ala Ser Ala Ala Ser Ala Ala145 150 155 160Ala Ser
Arg Leu Ser Ser Pro Glu Ala Ser Ser Arg Val Ser Ser Ala 165 170
175Val Ser Asn Leu Val Ser Ser Gly Pro Thr Asn Ser Ala Ala Leu Ser
180 185 190Ser Thr Ile Ser Asn Val Val Ser Gln Ile Gly Ala Ser Asn
Pro Gly 195 200 205Leu Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu
Glu Val Val Ser 210 215 220Ala Leu Ile His Ile Leu Gly Ser Ser Ser
Ile Gly Gln Val Asn Tyr225 230 235 240Gly Ser Ala Gly Gln Ala Thr
Gln Leu Val Gly Gln Ser Val Tyr Gln 245 250
255Ala13276PRTArtificial SequenceHybrid sequence derived from
Euprosthenops australis and Euprosthenops sp MaSp1 13Gly Ser Ala
Met Gly Gln Gly Ser Gly Gly Asn Ala Ala Ala Ala Ala1 5 10 15Ala Ala
Ala Ala Ala Ala Ala Ala Ser Gly Gln Gly Gly Gln Gly Gly 20 25 30Gln
Gly Gln Gly Gly Tyr Gly Gln Gly Ala Gly Ile Ser Ala Ala Ala 35 40
45Ala Ala Ala Ala Ala Ala Ala Ala Gly Ala Ala Ala Gly Arg Gly Gln
50 55 60Gly Gly Tyr Gly Gln Gly Ala Gly Gly Asn Ala Ala Ala Ala Ala
Ala65 70 75 80Ala Ala Ala Ala Ala Ala Ala Ala Gly Gln Gly Gly Gln
Gly Gly Tyr 85 90 95Gly Gly Gln Gly Leu Gly Gly Tyr Gly Gln Gly Ala
Gly Ser Ser Ala 100 105 110Ala Ala Ala Ala Ala Ala Ala Gly Arg Gly
Gln Ser Val Tyr Ala Ser 115 120 125Gly Gly Ala Gly Gln Gly Gly Tyr
Gly Gly Leu Gly Gly Gln Gly Ala 130 135 140Gly Arg Gly Gly Gln Gly
Ala Gly Ala Ala Ala Ala Ala Ala Gly Gly145 150 155 160Ser Gly Gln
Gly Gly Tyr Gly Gly Val Gly Ser Gly Ala Ser Ala Ala 165 170 175Ser
Ala Ala Ala Ser Arg Leu Ser Ser Pro Glu Ala Ser Ser Arg Val 180 185
190Ser Ser Ala Val Ser Asn Leu Val Ser Ser Gly Pro Thr Asn Ser Ala
195 200 205Ala Leu Ser Ser Thr Ile Ser Asn Val Val Ser Gln Ile Gly
Ala Ser 210 215 220Asn Pro Gly Leu Ser Gly Cys Asp Val Leu Val Gln
Ala Leu Leu Glu225 230 235 240Val Val Ser Ala Leu Ile His Ile Leu
Gly Ser Ser Ser Ile Gly Gln 245 250 255Val Asn Tyr Gly Ser Ala Gly
Gln Ala Thr Gln Leu Val Gly Gln Ser 260 265 270Val Tyr Gln Ala
27514269PRTArtificial SequenceSequence derived from Euprosthenops
australis MaSp1 14Gly Ser Ala Met Gly Tyr Leu Trp Ile Gln Gly Gln
Gly Gly Tyr Gly1 5 10 15Gly Leu Gly Gln Gly Gly Tyr Gly Gln Gly Ala
Gly Ser Ser Ala Ala 20 25 30Ala Ala Ala Cys Cys Ala Ala Ala Ala Ala
Gly Gly Gln Gly Gly Gln 35 40 45Gly Gln Gly Gly Tyr Gly Gln Gly Ser
Gly Gly Ser Ala Ala Ala Ala 50 55 60Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Gly Arg Gly Gln Gly65 70 75 80Gly Tyr Gly Gln Gly Ser
Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala 85 90 95Ala Ala Ala Ala Ala
Ala Ala Ala Gly Gln Gly Gly Gln Gly Gly Tyr 100 105 110Gly Arg Gln
Ser Gln Gly Ala Gly Ser Ala Ala Ala Ala Ala Ala Ala 115 120 125Ala
Ala Ala Ala Ala Ala Ala Gly Ser Gly Gln Gly Gly Tyr Gly Gly 130 135
140Gln Gly Gln Gly Gly Tyr Gly Gln Ser Ser Ala Ser Ala Ser Ala
Ala145 150 155 160Ala Ser Ala Ala Ser Thr Val Ala Asn Ser Val Ser
Arg Leu Ser Ser 165 170 175Pro Ser Ala Val Ser Arg Val Ser Ser Ala
Val Ser Ser Leu Val Ser 180 185 190Asn Gly Gln Val Asn Met Ala Ala
Leu Pro Asn Ile Ile Ser Asn Ile 195 200 205Ser Ser Ser Val Ser Ala
Ser Ala Pro Gly Ala Ser Gly Cys Glu Val 210 215 220Ile Val Gln Ala
Leu Leu Glu Val Ile Thr Ala Leu Val Gln Ile Val225 230 235 240Ser
Ser Ser Ser Val Gly Tyr Ile Asn Pro Ser Ala Val Asn Gln Ile 245 250
255Thr Asn Val Val Ala Asn Ala Met Ala Gln Val Met Gly 260
26515269PRTArtificial SequenceSequence derived from Euprosthenops
australis MaSp1 15Gly Ser Ala Met Gly Tyr Leu Trp Ile Gln Gly Gln
Gly Gly Tyr Gly1 5 10 15Gly Leu Gly Gln Gly Gly Tyr Gly Gln Gly Ala
Gly Ser Ser Ala Ala 20 25 30Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Gly Gly Gln Gly Gly Gln 35 40 45Gly Gln Gly Gly Tyr Gly Gln Gly Ser
Gly Gly Ser Ala Ala Ala Ala 50 55 60Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Gly Arg Gly Gln Gly65 70 75
80Gly Tyr Gly Gln Gly Ser Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala
85 90 95Ala Ala Ala Ala Ala Ala Ala Ala Gly Gln Gly Gly Gln Gly Gly
Tyr 100 105 110Gly Arg Gln Ser Gln Gly Ala Gly Ser Ala Ala Ala Ala
Ala Ala Cys 115 120 125Cys Ala Ala Ala Ala Ala Ala Gly Ser Gly Gln
Gly Gly Tyr Gly Gly 130 135 140Gln Gly Gln Gly Gly Tyr Gly Gln Ser
Ser Ala Ser Ala Ser Ala Ala145 150 155 160Ala Ser Ala Ala Ser Thr
Val Ala Asn Ser Val Ser Arg Leu Ser Ser 165 170 175Pro Ser Ala Val
Ser Arg Val Ser Ser Ala Val Ser Ser Leu Val Ser 180 185 190Asn Gly
Gln Val Asn Met Ala Ala Leu Pro Asn Ile Ile Ser Asn Ile 195 200
205Ser Ser Ser Val Ser Ala Ser Ala Pro Gly Ala Ser Gly Cys Glu Val
210 215 220Ile Val Gln Ala Leu Leu Glu Val Ile Thr Ala Leu Val Gln
Ile Val225 230 235 240Ser Ser Ser Ser Val Gly Tyr Ile Asn Pro Ser
Ala Val Asn Gln Ile 245 250 255Thr Asn Val Val Ala Asn Ala Met Ala
Gln Val Met Gly 260 26516269PRTArtificial SequenceSequence derived
from Euprosthenops australis MaSp1 16Gly Ser Ala Met Gly Tyr Leu
Trp Ile Gln Gly Gln Gly Gly Tyr Gly1 5 10 15Gly Leu Gly Gln Gly Gly
Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala 20 25 30Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Gly Gly Gln Gly Gly Gln 35 40 45Gly Gln Gly Gly
Tyr Gly Gln Gly Ser Gly Gly Ser Ala Ala Ala Ala 50 55 60Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly65 70 75 80Gly
Tyr Gly Gln Gly Ser Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala 85 90
95Ala Ala Ala Ala Ala Ala Ala Ala Gly Gln Gly Gly Gln Gly Gly Tyr
100 105 110Gly Arg Gln Ser Gln Gly Ala Gly Ser Ala Ala Ala Ala Ala
Ala Ala 115 120 125Ala Ala Ala Ala Ala Ala Ala Gly Ser Gly Gln Gly
Gly Tyr Gly Gly 130 135 140Gln Gly Gln Gly Gly Tyr Gly Gln Ser Ser
Ala Ser Ala Ser Ala Ala145 150 155 160Ala Ser Ala Ala Ser Thr Val
Ala Asn Ser Val Ser Arg Leu Ser Ser 165 170 175Pro Ser Ala Val Ser
Arg Val Ser Ser Ala Val Ser Ser Leu Val Ser 180 185 190Asn Gly Gln
Val Asn Met Ala Ala Leu Pro Asn Ile Ile Ser Asn Ile 195 200 205Ser
Ser Ser Val Ser Ala Ser Ala Pro Gly Ala Ser Gly Ser Glu Val 210 215
220Ile Val Gln Ala Leu Leu Glu Val Ile Thr Ala Leu Val Gln Ile
Val225 230 235 240Ser Ser Ser Ser Val Gly Tyr Ile Asn Pro Ser Ala
Val Asn Gln Ile 245 250 255Thr Asn Val Val Ala Asn Ala Met Ala Gln
Val Met Gly 260 26517384PRTArtificial SequenceSynthetic construct
17Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala1
5 10 15Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly 20 25 30Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly 35 40 45Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala 50 55 60Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly65 70 75 80Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly 85 90 95Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala 100 105 110Ala Ala Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly 115 120 125Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 130 135 140Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala145 150 155
160Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
165 170 175Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly 180 185 190Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala 195 200 205Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly 210 215 220Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly225 230 235 240Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 245 250 255Ala Ala Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 260 265 270Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 275 280
285Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
290 295 300Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly305 310 315 320Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly 325 330 335Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala 340 345 350Ala Ala Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly 355 360 365Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 370 375
38018402PRTArtificial SequenceSynthetic construct 18Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala1 5 10 15Ala Ala Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 20 25 30Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 35 40 45Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 50 55
60Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly65
70 75 80Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly 85 90 95Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala 100 105 110Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly 115 120 125Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly 130 135 140Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala145 150 155 160Ala Ala Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 165 170 175Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 180 185 190Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 195 200
205Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
210 215 220Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly225 230 235 240Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala 245 250 255Ala Ala Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly 260 265 270Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly 275 280 285Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 290 295 300Ala Ala Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly305 310 315
320Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
325 330 335Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala 340 345 350Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly 355 360 365Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly 370 375 380Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala385 390 395 400Ala
Ala19384PRTArtificial SequenceSynthetic construct 19Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly1 5 10 15Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Ala 20 25 30Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 35 40 45Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 50 55
60Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Ala65
70 75 80Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala 85 90 95Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly 100 105 110Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Ala Ala 115 120 125Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala 130 135 140Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly145 150 155 160Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Ala 165 170 175Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 180 185 190Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 195 200
205Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Ala
210 215 220Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala225 230 235 240Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly 245 250 255Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Ala Ala 260 265 270Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala 275 280 285Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 290 295 300Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Ala305 310 315
320Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
325 330 335Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly 340 345 350Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Ala Ala 355 360 365Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala 370 375 38020414PRTArtificial
SequenceSynthetic construct 20Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Ala Ala 20 25 30Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala 35 40 45Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 50 55 60Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Ala65 70 75 80Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 85 90 95Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 100 105
110Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Ala
115 120 125Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala 130 135 140Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly145 150 155 160Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Ala Ala 165 170 175Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala 180 185 190Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 195 200 205Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Ala 210 215 220Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala225 230
235 240Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly 245 250 255Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Ala Ala 260 265 270Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala 275 280 285Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly 290 295 300Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Ala Ala305 310 315 320Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 325 330 335Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 340 345
350Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Ala
355 360 365Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala 370 375 380Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly385 390 395 400Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly 405 4102114PRTArtificial SequenceSynthetic
peptide 21Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala1
5 102218PRTArtificial SequenceSynthetic peptide 22Ala Ser Ala Ser
Ala Ala Ala Ser Ala Ala Ser Thr Val Ala Asn Ser1 5 10 15Val
Ser239PRTArtificial SequenceSynthetic peptide 23Ala Ser Ala Ala Ser
Ala Ala Ala Ser1 524100PRTArtificial SequenceSynthetic peptide
24Ser Arg Leu Ser Ser Pro Glu Ala Ser Ser Arg Val Ser Ser Ala Val1
5 10 15Ser Asn Leu Val Ser Ser Gly Pro Thr Asn Ser Ala Ala Leu Ser
Ser 20 25 30Thr Ile Ser Asn Val Val Ser Gln Ile Gly Ala Ser Asn Pro
Gly Leu 35 40 45Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val
Val Ser Ala 50 55 60Leu Ile His Ile Leu Gly Ser Ser Ser Ile Gly Gln
Val Asn Tyr Gly65 70 75 80Ser Ala Gly Gln Ala Thr Gln Leu Val Gly
Gln Ser Val Tyr Gln Ala 85 90 95Leu Gly Glu Phe
1002598PRTArtificial SequenceSynthetic peptide 25Ser Arg Leu Ser
Ser Pro Ser Ala Val Ser Arg Val Ser Ser Ala Val1 5 10 15Ser Ser Leu
Val Ser Asn Gly Gln Val Asn Met Ala Ala Leu Pro Asn 20 25 30Ile Ile
Ser Asn Ile Ser Ser Ser Val Ser Ala Ser Ala Pro Gly Ala 35 40 45Ser
Gly Cys Glu Val Ile Val Gln Ala Leu Leu Glu Val Ile Thr Ala 50 55
60Leu Val Gln Ile Val Ser Ser Ser Ser Val Gly Tyr Ile Asn Pro Ser65
70 75 80Ala Val Asn Gln Ile Thr Asn Val Val Ala Asn Ala Met Ala Gln
Val 85 90 95Met Gly2699PRTArtificial SequenceSynthetic peptide
26Ser Arg Leu Ser Ser Pro Gly Ala Ala Ser Arg Val Ser Ser Ala Val1
5 10 15Thr Ser Leu Val Ser Ser Gly Gly Pro Thr Asn Ser Ala Ala Leu
Ser 20 25 30Asn Thr Ile Ser Asn Val Val Ser Gln Ile Ser Ser Ser Asn
Pro Gly 35 40 45Leu Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu
Ile Val Ser 50 55 60Ala Leu Val His Ile Leu Gly Ser Ala Asn Ile Gly
Gln Val Asn Ser65 70 75 80Ser Gly Val Gly Arg Ser Ala Ser Ile Val
Gly Gln Ser Ile Asn Gln 85 90 95Ala Phe Ser2789PRTArtificial
SequenceSynthetic peptide 27Ser His Leu Ser Ser Pro Glu Ala Ser Ser
Arg Val Ser Ser Ala Val1 5 10 15Ser Asn Leu Val Ser Ser Gly Ser Thr
Asn Ser Ala Ala Leu Pro Asn 20
25 30Thr Ile Ser Asn Val Val Ser Gln Ile Ser Ser Ser Asn Pro Gly
Leu 35 40 45Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val
Ser Ala 50 55 60Leu Ile His Ile Leu Gly Ser Ser Ser Ile Gly Gln Val
Asn Tyr Gly65 70 75 80Ser Ala Gly Gln Ala Thr Gln Ile Val
852898PRTArtificial SequenceSynthetic peptide 28Ser Ala Leu Ala Ala
Pro Ala Thr Ser Ala Arg Ile Ser Ser His Ala1 5 10 15Ser Thr Leu Leu
Ser Asn Gly Pro Thr Asn Pro Ala Ser Ile Ser Asn 20 25 30Val Ile Ser
Asn Ala Val Ser Gln Ile Ser Ser Ser Asn Pro Gly Ala 35 40 45Ser Ser
Cys Asp Val Leu Val Gln Ala Leu Leu Glu Leu Val Thr Ala 50 55 60Leu
Leu Thr Ile Ile Gly Ser Ser Asn Val Gly Asn Val Asn Tyr Asp65 70 75
80Ser Ser Gly Gln Tyr Ala Gln Val Val Ser Gln Ser Val Gln Asn Ala
85 90 95Phe Val2998PRTArtificial SequenceSynthetic peptide 29Ser
Ala Leu Ser Ala Pro Ala Thr Ser Ala Arg Ile Ser Ser His Ala1 5 10
15Ser Ala Leu Leu Ser Ser Gly Pro Thr Asn Pro Ala Ser Ile Ser Asn
20 25 30Val Ile Ser Asn Ala Val Ser Gln Ile Ser Ser Ser Asn Pro Gly
Ala 35 40 45Ser Ala Cys Asp Val Leu Val Gln Ala Leu Leu Glu Leu Val
Thr Ala 50 55 60Leu Leu Thr Ile Ile Gly Ser Ser Asn Ile Gly Ser Val
Asn Tyr Asp65 70 75 80Ser Ser Gly Gln Tyr Ala Gln Val Val Thr Gln
Ser Val Gln Asn Val 85 90 95Phe Gly3093PRTArtificial
SequenceSynthetic peptide 30Ser His Leu Ser Ser Pro Glu Ala Ser Ser
Arg Val Ser Ser Ala Val1 5 10 15Ser Asn Leu Val Ser Gly Gly Ser Thr
Asn Ser Ala Ala Leu Pro Asn 20 25 30Thr Ile Ser Asn Val Val Ser Gln
Ile Ser Ser Ser Asn Pro Gly Leu 35 40 45Ser Gly Cys Asp Val Leu Val
Gln Ala Leu Leu Glu Val Val Ser Ala 50 55 60Leu Ile His Ile Leu Gly
Ser Ser Ser Ile Gly Gln Val Asp Tyr Gly65 70 75 80Ser Ala Gly Gln
Ala Thr Gln Ile Val Gly Gln Ser Ala 85 903198PRTArtificial
SequenceSynthetic peptide 31Ser Arg Leu Ser Ser Pro Gln Ala Ser Ser
Arg Val Ser Ser Ala Val1 5 10 15Ser Asn Leu Val Ala Ser Gly Pro Thr
Asn Ser Ala Ala Leu Ser Ser 20 25 30Thr Ile Ser Asn Val Val Ser Gln
Ile Gly Ala Ser Asn Pro Gly Leu 35 40 45Ser Gly Cys Asp Val Leu Ile
Gln Ala Leu Leu Glu Val Val Ser Ala 50 55 60Leu Ile Gln Ile Leu Gly
Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly65 70 75 80Ser Ala Gly Gln
Ala Thr Gln Ile Val Gly Gln Ser Val Tyr Gln Ala 85 90 95Leu
Gly3289PRTArtificial SequenceSynthetic peptide 32Ser Arg Leu Ser
Ser Pro Glu Ala Ser Ser Arg Val Ser Ser Ala Val1 5 10 15Ser Asn Leu
Val Ser Ser Gly Pro Thr Asn Ser Ala Ala Leu Ser Asn 20 25 30Thr Ile
Ser Asn Val Val Ser Gln Ile Ser Ser Ser Asn Pro Gly Leu 35 40 45Ser
Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala 50 55
60Leu Ile His Ile Leu Gly Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly65
70 75 80Ser Ala Gly Gln Ala Thr Gln Ile Val 853387PRTArtificial
SequenceSynthetic peptide 33Ser Arg Leu Ser Ser Pro Gln Ala Ser Ser
Arg Val Ser Ser Ala Val1 5 10 15Ser Asn Leu Val Ala Ser Gly Pro Thr
Asn Ser Ala Ala Leu Ser Ser 20 25 30Thr Ile Ser Asn Ala Val Ser Gln
Ile Gly Ala Ser Asn Pro Gly Leu 35 40 45Ser Gly Cys Asp Val Leu Ile
Gln Ala Leu Leu Glu Val Val Ser Ala 50 55 60Leu Ile His Ile Leu Gly
Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly65 70 75 80Ser Ala Gly Gln
Ala Thr Gln 853487PRTArtificial SequenceSynthetic peptide 34Ser Arg
Leu Ser Ser Pro Glu Ala Ser Ser Arg Val Ser Ser Ala Val1 5 10 15Ser
Asn Leu Val Ser Ser Gly Pro Thr Asn Ser Ala Ala Leu Ser Ser 20 25
30Thr Ile Ser Asn Val Val Ser Gln Ile Gly Ala Ser Asn Pro Gly Leu
35 40 45Ser Gly Cys Asp Val Leu Ile Gln Ala Leu Leu Glu Val Val Ser
Ala 50 55 60Leu Val His Ile Leu Gly Ser Ser Ser Ile Gly Gln Val Asn
Tyr Gly65 70 75 80Ser Ala Gly Gln Ala Thr Gln 853589PRTArtificial
SequenceSynthetic peptide 35Ser Arg Leu Ser Ser Pro Glu Ala Ser Ser
Arg Val Ser Ser Ala Val1 5 10 15Ser Asn Leu Val Ser Ser Gly Pro Thr
Asn Ser Ala Ala Leu Ser Asn 20 25 30Thr Ile Ser Asn Val Val Ser Gln
Ile Ser Ser Ser Asn Pro Gly Leu 35 40 45Ser Gly Cys Asp Val Leu Val
Gln Ala Leu Leu Glu Val Val Ser Ala 50 55 60Pro Ile His Ile Leu Gly
Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly65 70 75 80Ser Ala Gly Gln
Ala Thr Gln Ile Val 853689PRTArtificial SequenceSynthetic peptide
36Ser Arg Leu Ser Ser Pro Glu Ala Ser Ser Arg Val Ser Ser Ala Val1
5 10 15Ser Asn Leu Val Ser Ser Gly Pro Thr Asn Ser Ala Ala Leu Pro
Asn 20 25 30Thr Ile Ser Asn Val Val Ser Gln Ile Ser Ser Ser Asn Pro
Gly Leu 35 40 45Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val
Val Ser Ala 50 55 60Leu Ile His Ile Leu Gly Ser Ser Ser Ile Gly Gln
Val Asn Tyr Gly65 70 75 80Ser Ala Gly Gln Ala Thr Gln Ile Val
853788PRTArtificial SequenceSynthetic peptide 37Ser Leu Leu Ser Ser
Pro Ala Ser Asn Ala Arg Ile Ser Ser Ala Val1 5 10 15Ser Ala Leu Ala
Ser Gly Ala Ala Ser Gly Pro Gly Tyr Leu Ser Ser 20 25 30Val Ile Ser
Asn Val Val Ser Gln Val Ser Ser Asn Ser Gly Gly Leu 35 40 45Val Gly
Cys Asp Thr Leu Val Gln Ala Leu Leu Glu Ala Ala Ala Ala 50 55 60Leu
Val His Val Leu Ala Ser Ser Ser Gly Gly Gln Val Asn Leu Asn65 70 75
80Thr Ala Gly Tyr Thr Ser Gln Leu 853888PRTArtificial
SequenceSynthetic peptide 38Ser Arg Leu Ser Ser Pro Ala Ser Asn Ala
Arg Ile Ser Ser Ala Val1 5 10 15Ser Ala Leu Ala Ser Gly Gly Ala Ser
Ser Pro Gly Tyr Leu Ser Ser 20 25 30Ile Ile Ser Asn Val Val Ser Gln
Val Ser Ser Asn Asn Asp Gly Leu 35 40 45Ser Gly Cys Asp Thr Val Val
Gln Ala Leu Leu Glu Val Ala Ala Ala 50 55 60Leu Val His Val Leu Ala
Ser Ser Asn Ile Gly Gln Val Asn Leu Asn65 70 75 80Thr Ala Gly Tyr
Thr Ser Gln Leu 853989PRTArtificial SequenceSynthetic peptide 39Ser
Arg Leu Ser Ser Ser Ala Ala Ser Ser Arg Val Ser Ser Ala Val1 5 10
15Ser Ser Leu Val Ser Ser Gly Pro Thr Thr Pro Ala Ala Leu Ser Asn
20 25 30Thr Ile Ser Ser Ala Val Ser Gln Ile Ser Ala Ser Asn Pro Gly
Leu 35 40 45Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val
Ser Ala 50 55 60Leu Val His Ile Leu Gly Ser Ser Ser Val Gly Gln Ile
Asn Tyr Gly65 70 75 80Ala Ser Ala Gln Tyr Ala Gln Met Val
854097PRTArtificial SequenceSynthetic peptide 40Arg Leu Ser Ser Pro
Gln Ala Ser Ser Arg Val Ser Ser Ala Val Ser1 5 10 15Thr Leu Val Ser
Ser Gly Pro Thr Asn Pro Ala Ser Leu Ser Asn Ala 20 25 30Ile Gly Ser
Val Val Ser Gln Val Ser Ala Ser Asn Pro Gly Leu Pro 35 40 45Ser Cys
Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu 50 55 60Val
His Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ser Ala65 70 75
80Ser Ser Gln Tyr Ala Arg Leu Val Gly Gln Ser Ile Ala Gln Ala Leu
85 90 95Gly4182PRTArtificial SequenceSynthetic peptide 41Ser Arg
Leu Ser Ser Pro Gln Ala Ser Ser Arg Val Ser Ser Ala Val1 5 10 15Ser
Thr Leu Val Ser Ser Gly Pro Thr Asn Pro Ala Ala Leu Ser Asn 20 25
30Ala Ile Ser Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro Gly Leu
35 40 45Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Leu Val Ser
Ala 50 55 60Leu Val His Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn
Tyr Ala65 70 75 80Ala Ser4298PRTArtificial SequenceSynthetic
peptide 42Ser Arg Leu Ser Ser Pro Gln Ala Ser Ser Arg Val Ser Ser
Ala Val1 5 10 15Ser Thr Leu Val Ser Ser Gly Pro Thr Asn Pro Ala Ser
Leu Ser Asn 20 25 30Ala Ile Ser Ser Val Val Ser Gln Val Ser Ser Ser
Asn Pro Gly Leu 35 40 45Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu
Glu Ile Val Ser Ala 50 55 60Leu Val His Ile Leu Gly Ser Ser Ser Ile
Gly Gln Ile Asn Tyr Ala65 70 75 80Ala Ser Ser Gln Tyr Ala Gln Leu
Val Gly Gln Ser Leu Thr Gln Ala 85 90 95Leu Gly4389PRTArtificial
SequenceSynthetic peptide 43Ser Arg Leu Ser Ser Pro Gln Ala Gly Ala
Arg Val Ser Ser Ala Val1 5 10 15Ser Ala Leu Val Ala Ser Gly Pro Thr
Ser Pro Ala Ala Val Ser Ser 20 25 30Ala Ile Ser Asn Val Ala Ser Gln
Ile Ser Ala Ser Asn Pro Gly Leu 35 40 45Ser Gly Cys Asp Val Leu Val
Gln Ala Leu Leu Glu Ile Val Ser Ala 50 55 60Leu Val Ser Ile Leu Ser
Ser Ala Ser Ile Gly Gln Ile Asn Tyr Gly65 70 75 80Ala Ser Gly Gln
Tyr Ala Ala Met Ile 854490PRTArtificial SequenceSynthetic peptide
44Ser Ala Leu Ser Ser Pro Thr Thr His Ala Arg Ile Ser Ser His Ala1
5 10 15Ser Thr Leu Leu Ser Ser Gly Pro Thr Asn Ser Ala Ala Ile Ser
Asn 20 25 30Val Ile Ser Asn Ala Val Ser Gln Val Ser Ala Ser Asn Pro
Gly Ser 35 40 45Ser Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Leu
Ile Thr Ala 50 55 60Leu Ile Ser Ile Val Asp Ser Ser Asn Ile Gly Gln
Val Asn Tyr Gly65 70 75 80Ser Ser Gly Gln Tyr Ala Gln Met Val Gly
85 904598PRTArtificial SequenceSynthetic peptide 45Ser Ala Leu Ser
Ser Pro Thr Thr His Ala Arg Ile Ser Ser His Ala1 5 10 15Ser Thr Leu
Leu Ser Ser Gly Pro Thr Asn Ala Ala Ala Leu Ser Asn 20 25 30Val Ile
Ser Asn Ala Val Ser Gln Val Ser Ala Ser Asn Pro Gly Ser 35 40 45Ser
Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Ile Thr Ala 50 55
60Leu Ile Ser Ile Leu Asp Ser Ser Ser Val Gly Gln Val Asn Tyr Gly65
70 75 80Ser Ser Gly Gln Tyr Ala Gln Ile Val Gly Gln Ser Met Gln Gln
Ala 85 90 95Met Gly4697PRTArtificial SequenceSynthetic peptide
46Ser Arg Leu Ala Ser Pro Asp Ser Gly Ala Arg Val Ala Ser Ala Val1
5 10 15Ser Asn Leu Val Ser Ser Gly Pro Thr Ser Ser Ala Ala Leu Ser
Ser 20 25 30Val Ile Ser Asn Ala Val Ser Gln Ile Gly Ala Ser Asn Pro
Gly Leu 35 40 45Ser Gly Cys Asp Val Leu Ile Gln Ala Leu Leu Glu Ile
Val Ser Ala 50 55 60Cys Val Thr Ile Leu Ser Ser Ser Ser Ile Gly Gln
Val Asn Tyr Gly65 70 75 80Ala Ala Ser Gln Phe Ala Gln Val Val Gly
Gln Ser Val Leu Ser Ala 85 90 95Phe4782PRTArtificial
SequenceSynthetic peptide 47Ser Arg Leu Ala Ser Pro Asp Ser Gly Ala
Arg Val Ala Ser Ala Val1 5 10 15Ser Asn Leu Val Ser Ser Gly Pro Thr
Ser Ser Ala Ala Leu Ser Ser 20 25 30Val Ile Ser Asn Ala Val Ser Gln
Ile Gly Ala Ser Asn Pro Gly Leu 35 40 45Ser Gly Cys Asp Val Leu Ile
Gln Ala Leu Leu Glu Ile Val Ser Ala 50 55 60Cys Val Thr Ile Leu Ser
Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly65 70 75 80Ala
Ala4882PRTArtificial SequenceSynthetic peptide 48Ser Arg Leu Ala
Ser Pro Asp Ser Gly Ala Arg Val Ala Ser Ala Val1 5 10 15Ser Asn Leu
Val Ser Ser Gly Pro Thr Ser Ser Ala Ala Leu Ser Ser 20 25 30Val Ile
Xaa Asn Ala Val Ser Gln Ile Gly Ala Ser Asn Pro Gly Leu 35 40 45Ser
Gly Cys Asp Val Leu Leu Xaa Ala Leu Leu Glu Ile Val Ser Ala 50 55
60Cys Val Thr Ile Leu Ser Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly65
70 75 80Ala Ala4971PRTArtificial SequenceSynthetic peptide 49Ser
Arg Leu Ser Ser Pro Glu Ala Ala Ser Arg Val Ser Ser Ala Val1 5 10
15Ser Ser Leu Val Ser Asn Gly Gln Val Asn Val Asp Ala Leu Pro Ser
20 25 30Ile Ile Ser Asn Leu Ser Ser Ser Ile Ser Ala Ser Ala Thr Thr
Ala 35 40 45Ser Asp Cys Glu Val Leu Val Gln Val Leu Leu Glu Val Val
Ser Ala 50 55 60Leu Val Gln Ile Val Cys Ser65 705097PRTArtificial
SequenceSynthetic peptide 50Ser Arg Leu Ser Ser Pro Gln Ala Ala Ser
Arg Val Ser Ser Ala Val1 5 10 15Ser Ser Leu Val Ser Asn Gly Gln Val
Asn Val Ala Ala Leu Pro Ser 20 25 30Ile Ile Ser Ser Leu Ser Ser Ser
Ile Ser Ala Ser Ser Thr Ala Ala 35 40 45Ser Asp Cys Glu Val Leu Val
Gln Val Leu Leu Glu Ile Val Ser Ala 50 55 60Leu Val Gln Ile Val Ser
Ser Ala Asn Val Gly Tyr Ile Asn Pro Glu65 70 75 80Ala Ser Gly Ser
Leu Asn Ala Val Gly Ser Ala Leu Ala Ala Ala Met 85 90
95Gly5193PRTArtificial SequenceSynthetic peptide 51Asn Arg Leu Ser
Ser Ala Gly Ala Ala Ser Arg Val Ser Ser Asn Val1 5 10 15Ala Ala Ile
Ala Ser Ala Gly Ala Ala Ala Leu Pro Asn Val Ile Ser 20 25 30Asn Ile
Tyr Ser Gly Val Leu Ser Ser Gly Val Ser Ser Ser Glu Ala 35 40 45Leu
Ile Gln Ala Leu Leu Glu Val Ile Ser Ala Leu Ile His Val Leu 50 55
60Gly Ser Ala Ser Ile Gly Asn Val Ser Ser Val Gly Val Asn Ser Ala65
70 75 80Leu Asn Ala Val Gln Asn Ala Val Gly Ala Tyr Ala Gly 85
905298PRTArtificial SequenceSynthetic peptide 52Ser Arg Leu Ser Ser
Pro Ser Ala Ala Ala Arg Val Ser Ser Ala Val1 5 10 15Ser Leu Val Ser
Asn Gly Gly Pro Thr Ser Pro Ala Ala Leu Ser Ser 20 25 30Ser Ile Ser
Asn Val Val Ser Gln Ile Ser Ala Ser Asn Pro Gly Leu 35 40 45Ser Gly
Cys Asp Ile Leu Val Gln Ala Leu Leu Glu Leu Ile Ser Ala 50 55 60Leu
Val His Ile Leu Gly Ser Ala Asn Ile Gly Pro Val Asn Ser Ser65 70 75
80Ser Ala Gly Gln Ser Ala Ser Ile Val Gly Gln Ser Val Tyr Arg Ala
85 90 95Leu Ser5398PRTArtificial SequenceSynthetic peptide 53Ser
Arg Leu Ser Ser Pro Ala Ala Ser Ser Arg Val Ser Ser Ala
Val1 5 10 15Ser Ser Leu Val Ser Ser Gly Pro Thr Lys His Ala Ala Leu
Ser Asn 20 25 30Thr Ile Ser Ser Val Val Ser Gln Val Ser Ala Ser Asn
Pro Gly Leu 35 40 45Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu
Val Val Ser Ala 50 55 60Leu Val Ser Ile Leu Gly Ser Ser Ser Ile Gly
Gln Ile Asn Tyr Gly65 70 75 80Ala Ser Ala Gln Tyr Thr Gln Met Val
Gly Gln Ser Val Ala Gln Ala 85 90 95Leu Ala5494PRTArtificial
SequenceSynthetic peptide 54Ser Val Tyr Leu Arg Leu Gln Pro Arg Leu
Glu Val Ser Ser Ala Val1 5 10 15Ser Ser Leu Val Ser Ser Gly Pro Thr
Asn Gly Ala Ala Val Ser Gly 20 25 30Ala Leu Asn Ser Leu Val Ser Gln
Ile Ser Ala Ser Asn Pro Gly Leu 35 40 45Ser Gly Cys Asp Ala Leu Val
Gln Ala Leu Leu Glu Leu Val Ser Ala 50 55 60Leu Val Ala Ile Leu Ser
Ser Ala Ser Ile Gly Gln Val Asn Val Ser65 70 75 80Ser Val Ser Gln
Ser Thr Gln Met Ile Ser Gln Ala Leu Ser 85 9055100PRTArtificial
SequenceSynthetic peptide 55Ser Arg Leu Ser Ser Pro Gln Ala Ser Ser
Arg Val Ser Ser Ala Val1 5 10 15Ser Asn Leu Val Ser Ser Gly Pro Thr
Asn Ser Ala Ala Leu Ser Asn 20 25 30Thr Ile Ser Asn Val Val Ser Gln
Leu Ser Ala Ser Asn Pro Gly Leu 35 40 45Ser Gly Cys Asp Val Leu Val
Gln Ala Leu Leu Glu Val Val Ser Ala 50 55 60Leu Val His Ile Leu Gly
Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly65 70 75 80Ser Ala Gly Gln
Ala Thr Gln Ile Val Gly Gln Ser Val Ala Gln Ala 85 90 95Leu Gly Glu
Phe 10056240PRTArtificial SequenceSynthetic construct 56Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala1 5 10 15Ala Ala
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 20 25 30Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 35 40
45Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
50 55 60Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly65 70 75 80Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly 85 90 95Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala 100 105 110Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly 115 120 125Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly 130 135 140Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala145 150 155 160Ala Ala Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 165 170 175Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 180 185
190Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
195 200 205Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly 210 215 220Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly225 230 235 24057258PRTArtificial SequenceSynthetic
construct 57Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala1 5 10 15Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly 20 25 30Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly 35 40 45Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala 50 55 60Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly65 70 75 80Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly 85 90 95Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala 100 105 110Ala Ala Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 115 120 125Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 130 135 140Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala145 150
155 160Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly 165 170 175Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly 180 185 190Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala 195 200 205Ala Ala Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly 210 215 220Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly225 230 235 240Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 245 250 255Ala
Ala58240PRTArtificial SequenceSynthetic construct 58Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly1 5 10 15Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Ala 20 25 30Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 35 40 45Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 50 55
60Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Ala65
70 75 80Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala 85 90 95Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly 100 105 110Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Ala Ala 115 120 125Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala 130 135 140Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly145 150 155 160Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Ala 165 170 175Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 180 185 190Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 195 200
205Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Ala
210 215 220Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala225 230 235 24059270PRTArtificial SequenceSynthetic
construct 59Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Ala Ala 20 25 30Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala 35 40 45Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly 50 55 60Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Ala Ala65 70 75 80Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala 85 90 95Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly 100 105 110Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Ala 115 120 125Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 130 135 140Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly145 150
155 160Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala
Ala 165 170 175Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala 180 185 190Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly 195 200 205Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Ala Ala 210 215 220Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala225 230 235 240Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 245 250 255Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 260 265
2706018PRTArtificial SequenceSynthetic peptide 60Ala Ser Ala Ser
Ala Ala Ala Ser Ala Ala Ser Thr Val Ala Asn Ser1 5 10 15Val
Ser618PRTArtificial SequenceSynthetic peptide 61Ala Ser Ala Ala Ser
Ala Ala Ala1 5628PRTArtificial SequenceSynthetic peptide 62Gly Ser
Ala Met Gly Gln Gly Ser1 5636PRTArtificial SequenceSynthetic
peptide 63Leu Val Pro Arg Gly Ser1 5644PRTArtificial
SequenceSynthetic peptide 64Asp Asp Asp Lys1658PRTArtificial
SequenceSynthetic peptide 65Leu Gly Val Leu Phe Gln Gly Pro1
5664PRTArtificial SequenceSynthetic peptide 66Ile Xaa Gly
Arg1677PRTArtificial SequenceSynthetic peptide 67Glu Xaa Xaa Tyr
Xaa Gln Xaa1 5687PRTArtificial SequenceSynthetic peptide 68Glu Asn
Leu Tyr Phe Gln Gly1 5698PRTArtificial SequenceSynthetic peptide
69Glu Asp Asn Leu Tyr Phe Gln Gly1 5708PRTArtificial
SequenceSynthetic peptide 70Leu Glu Val Leu Phe Gln Gly Pro1
5715PRTArtificial SequenceSynthetic peptide 71Gly Arg Gly Gln Gly1
5725PRTArtificial SequenceSynthetic peptide 72Gly Gln Gly Gln Gly1
5
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