U.S. patent application number 10/924092 was filed with the patent office on 2005-06-23 for mutant proteins having lower allergenic response in humans and methods for constructing, identifying and producing such proteins.
Invention is credited to Estell, David A., Harding, Fiona A..
Application Number | 20050137112 10/924092 |
Document ID | / |
Family ID | 22032273 |
Filed Date | 2005-06-23 |
United States Patent
Application |
20050137112 |
Kind Code |
A1 |
Estell, David A. ; et
al. |
June 23, 2005 |
Mutant proteins having lower allergenic response in humans and
methods for constructing, identifying and producing such
proteins
Abstract
The present invention relates to a novel improved protein mutant
which produces low allergenic response in humans compared to the
parent of that mutant. Specifically, the present invention
comprises neutralizing or reducing the ability of T-cells to
recognize epitopes and thus prevent sensitization of an individual
to the protein.
Inventors: |
Estell, David A.; (San
Mateo, CA) ; Harding, Fiona A.; (Santa Clara,
CA) |
Correspondence
Address: |
Genencor International, Inc.
925 Page Mill Road
Palo Alto
CA
94304-1013
US
|
Family ID: |
22032273 |
Appl. No.: |
10/924092 |
Filed: |
August 23, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10924092 |
Aug 23, 2004 |
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09060872 |
Apr 15, 1998 |
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6835550 |
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Current U.S.
Class: |
510/320 ;
435/220; 435/252.31; 435/471; 435/69.1; 536/23.2 |
Current CPC
Class: |
A61P 37/08 20180101;
C12N 9/54 20130101; G01N 33/505 20130101; G01N 33/6878
20130101 |
Class at
Publication: |
510/320 ;
435/220; 435/252.31; 435/069.1; 435/471; 536/023.2 |
International
Class: |
C11D 003/386; C07H
021/04; C12N 009/52; C12N 009/54; C12N 015/74 |
Claims
1-11. (canceled)
12. A method for determining T-cell epitopes in a protein of
interest comprising the steps of: (a) obtaining from a single blood
source a solution of dendritic cells and a solution of naive CD4+
T-cells; (b) promoting differentiation in said solution of
dendritic cells, by exposing said dendritic cells to at least one
cytokine; (c) combining said solution of differentiated dendritic
cells and said naive CD4+ T-cells with a peptide of interest; (d)
measuring the proliferation of said T-cells in said step (c).
13. A method of reducing the immunogenicity of a protein comprising
the steps of: (a) identifying at least one T-cell epitope in said
protein, using the method of claim 12; (b) modifying said protein
to neutralize said at least one T-cell epitope.
14. The method according to claim 13, wherein said epitope is
modified by: P1 (a) substituting the amino acid sequence of the
epitope with an analogous sequence from a homolog of the protein of
interest wherein said analogous sequence produces a lower response
from said T-cells than that of the protein of interest; or (b)
substituting the amino acid sequence of the epitope with a sequence
which substantially mimics the major tertiary structure attributes
of the epitope, but which produces a lowere response from T-cells
than that of the protein of interest.
15. A protein having reduced immunogenicity produced using the
method of claim 14.
16. A protein having reduced immunogenicity, wherein said protein
comprises a modification comprising the substitution or deletion of
amino acid residues which are identified as being within a T-cell
epitope identified using the method of claim 13.
Description
BACKGROUND OF THE INVENTION
[0001] A. Field of the Invention
[0002] The present invention relates to proteins which produce
lower allergenic response in humans exposed to such proteins, and
an assay predictive of such response. More specifically, the
present invention relates to a novel improved protein mutant which
produces very low allergenic response in humans sensitized to that
protein through exposure compared to the precursor of such protein
mutant.
[0003] B. State of the Art
[0004] Proteins used in industrial, pharmaceutical and commercial
applications are of increasing prevalence. As a result, the
increased exposure due to this prevalence has been responsible for
some safety hazards caused by the sensitization of certain persons
to those peptides, whereupon subsequent exposure causes extreme
allergic reactions which can be injurious and even fatal. For
example, proteases are known to cause dangerous hypersensitivity in
some individuals. As a result, despite the usefulness of proteases
in industry, e.g., in laundry detergents, cosmetics, textile
treatment etc. . . . , and the extensive research performed in the
field to provide improved proteases which have, for example, more
effective stain removal under detergency conditions, the use of
proteases in industry has been problematic due to their ability to
produce a hypersensitive allergenic response in some humans.
[0005] Much work has been done to alleviate these problems. Among
the strategies explored to reduce immunogenic potential of protease
use have been improved production processes which reduce potential
contact by controlling and minimizing workplace concentrations of
dust particles or aerosol carrying airborne protease, improved
granulation processes which reduce the amount of dust or aerosol
actually produced from the protease product, and improved recovery
processes to reduce the level of potentially allergenic
contaminants in the final product. However, efforts to reduce the
allergenicity of protease, per se, have, been relatively
unsuccessful. Alternatively, efforts have been made to mask
epitopes in protease which are recognized by immunoglobulin E (IgE)
in hypersensitive individuals (PCT Publication No. WO 92/10755) or
to enlarge or change the nature of the antigenic determinants by
attaching polymers or peptides/proteins to the problematic
protease.
[0006] When an adaptive immune response occurs in an exaggerated or
inappropriate form, the individual experiencing the reaction is
said to be hypersensitive. Hypersensitivity reactions are the
result of normally beneficial immune responses acting
inappropriately and sometimes cause inflammatory reactions and
tissue damage. They can be provoked by many antigens; and the cause
of a hypersensitivity reaction will vary from one individual to the
next. Hypersensitivity does not normally manifest itself upon first
contact with the antigen, but usually appears upon subsequent
contact. One form of hypersensitivity occurs when an IgE response
is directed against innocuous environmental antigens, such as
pollen, dust-mites or animal dander. The resulting release of
pharmacological mediators by IgE-sensitized mast cells produces an
acute inflammatory reaction with symptoms such as asthma or
rhinitis.
[0007] Nonetheless, a strategy comprising modifying the. IgE sites
will not generally be successful in preventing the cause of the
initial sensitization reaction. Accordingly, such strategies, while
perhaps neutralizing or reducing the severity of the subsequent
hypersensitivity reaction, will not reduce the number or persons
actually sensitized. For example, when a person is known to be
hypersensitive to a certain antigen, the general, and only safe,
manner of dealing with such a situation is to isolate the
hypersensitive person from the antigen as completely as possible.
Indeed, any other course of action would be dangerous to the health
of the hypersensitive individual. Thus, while reducing the danger
of a specific protein for a hypersensitive individual is important,
for industrial purposes it would be far more valuable to render a
protein incapable of initiating the hypersensitivity reaction in
the first place.
[0008] T-lymphocytes (T-cells) are key players in the induction and
regulation of immune responses and in the execution of
immunological effector functions. Specific immunity against
infectious agents and tumors is known to be dependent on these
cells and they are believed to contribute to the healing of
injuries. On the other hand, failure to control these responses can
lead to auto aggression. In general, antigen is presented to
T-cells in the form of antigen presenting cells which, through a
variety of cell surface mechanisms, capture and display antigen or
partial antigen in a manner suitable for antigen recognition by the
T-cell. Upon recognition of a specific epitope by the receptors on
the surface of the T-cells (T-cell receptors), the T-cells begin a
series of complex interactions, including proliferation, which
result in the production of antibody by B-cells. While T-cells and
B-cells are both activated by antigenic epitopes which exist on a
given protein or peptide, the actual epitopes recognized by these
mononuclear cells are generally not identical. In fact, the epitope
which activates a T-cell to initiate the creation of immunologic
diversity is quite often not the same epitope which is later
recognized by B-cells in the course of the immunologic response.
Thus, with respect to hypersensitivity, while the specific
antigenic interaction between the T-cell and the antigen is a
critical element in the initiation of the immune response to
antigenic exposure, the specifics of that interaction, i.e., the
epitope recognized, is often not relevant to subsequent development
of a full blown allergic reaction.
[0009] PCT Publication No. WO 96/40791 discloses a process for
producing polyalkylene oxide-polypeptide conjugates with reduced
allergenicity using polyalkylene oxide as a starting material.
[0010] PCT Publication No. WO 97/30148 discloses a polypeptide
conjugate with reduced allergenicity which comprises one polymeric
carrier molecule having two or more polypeptide molecules coupled
covalently thereto.
[0011] PCT Publication No. WO 96/17929 discloses a process for
producing polypeptides with reduced allergenicity comprising the
step of conjugating from 1 to 30 polymolecules to a parent
polypeptide.
[0012] PCT Publication No. WO 92/10755 discloses a method of
producing protein variants evoking a reduced immunogenic response
in animals. In this application, the proteins of interest, a series
of proteases and variants thereof; were used to immunized rats. The
sera from the rats was then used to measure the reactivity of the
polyclonal-antibodies already produced and present in the immunized
sera to the protein of interest and variants thereof. From these
results, it was possible to determine whether the antibodies in the
preparation were comparatively more or less reactive with the
protein and its variants, thus permitting an analysis of which
changes in the protein are likely to neutralize or reduce the
ability of the Ig to bind. From these tests on rats, the conclusion
was arrived at that changing any of subtilisin 309 residues
corresponding to 127, 128, 129, 130, 131,151, 136, 151, 152, 153,
154, 161, 162, 163,167, 168, 169, 170, 171, 172, 173,174, 175, 176,
186, 193, 194, 195, 196, 197, 247, 251, 261 will result in a change
in the immunological potential.
[0013] PCT Publication No. WO 94/10191 discloses low allergenic
proteins comprising oligomeric forms of the parent monomeric
protein, wherein the oligomer has substantially retained its
activity.
[0014] The prior art has provided methods of reducing the
allergenicity of certain proteins and identification of epitopes
which cause allergic reactions in some individuals, the assays used
to identify these epitopes generally involving measurement of IgE
and IgG antibody in blood sera previously exposed to the antigen.
Nonetheless, once an Ig reaction has been initiated, sensitization
has already occurred. Accordingly, there is a need for a method of
determining epitopes which cause sensitization in the first place,
as neutralization of these epitopes will result in significantly
less possibility for sensitization to occur, thus reducing the
possibility of initial sensitization.
SUMMARY OF THE INVENTION
[0015] It is an object of the invention to provide a protein having
decreased potential to cause allergenic response in humans compared
to a precursor protein.
[0016] It is a further object of the present invention to provide
for a protease variant which has useful activity in common protease
applications, such as detergents and or the treatment of wool to
prevent felting, in bar or liquid soap applications, dishcare
formulations, contact lens cleaning solutions or products, peptide
hydrolysis, waste treatment, textile applications such as
anti-felting, in cosmetic formulations and for skin care, or as
fusion-cleavage enzymes in protein production, which protease
variant can be more safely used due to its lowered allergenic
potential.
[0017] According to the present invention, a method for identifying
T-cell epitopes within a protein is provided. The present invention
provides an assay which identifies epitopes as follows: antigen
presenting cells are combined with nave human T-cells and with a
peptide of interest. In a preferred embodiment of the invention, a
method is provided wherein a T-cell epitope is recognized
comprising the steps of: (a) obtaining from a single blood source a
solution of dendritic cells and a solution of naive CD4+ and/or
CD8+ T-cells; (b) promoting differentiation in said solution of
dendritic cells; (c) combining said solution of differentiated
dendritic cells and said naive CD4+ and/or CD8+ T-cells with a
peptide of interest; (d) measuring the proliferation of T-cells in
said step (c).
[0018] According to another embodiment of the present invention, a
protein is provided in which a T-cell epitope is modified so as to
reduce or preferably neutralize., (eliminate) the ability of the
T-cell to identify that epitope. Thus, a protein is provided having
reduced allergenicity, wherein said protein comprises a
modification comprising the substitution or deletion of amino acid
residues which are identified as within a T-cell epitope. According
to a preferred embodiment, an epitope is determined in a protein or
peptide which, when recognized by a T-cell, results in the
proliferation of T-cells which is greater than the baseline. That
T-cell epitope is then modified so that, when the peptide
comprising the epitope is analyzed in the assay of the invention,
it results in lesser proliferation than the protein comprising the
unmodified epitope. More preferably, the epitope to be modified
produces greater than three times the baseline T-cell proliferation
in a sample. When modified, the epitope produces less than three
times the baseline T-cell proliferation, preferably less than two
times the baseline T-cell proliferation and most preferably less
than or substantially equal to the baseline T-cell proliferation in
a sample.
[0019] Preferably, the epitope is modified in one of the following
ways: (a) the amino acid sequence of the epitope is substituted
with an analogous sequence from a human homolog to the protein of
interest, i.e., human subtilisin or another human protease derived
subtilisin like molecule such as furin or the kexins (see e.g.,
Methods in Enzymology, Vol. 244., (1994) pp. 175 et seq; Roebroek
et al., EMBO J., Vol. 5, No. 9, pp. 2197-2202 (1986); Tomkinson et
al., Biochem., Vol. 30, pp.168-174 (1991); Keifer et al., DNA and
Cell Biol., Vol.10, No. 10, pp. 757-769 (1991)); (b) the amino acid
sequence of the epitope is substituted with an analogous sequence
from a non-human homolog to the protein of interest, which
analogous: sequence produces a lesser allergenic response due to
T-cell recognition-than that of the protein of interest; (c) the
amino acid sequence of the epitope is substituted with a sequence
which substantially mimics the major tertiary structure attributes
of the epitope, but which produces a lesser allergenic response due
to T-cell recognition than that of the protein of interest; or (d)
with any sequence which produces lesser allergenic response due to
T-cell recognition than that of the protein of interest.
[0020] In a specific embodiment of the invention, a protease
variant is provided comprising at least one amino acid substitution
at a position corresponding to residues 170, 171, 172 and/or 173 in
BPN', wherein such substitutions comprise modifying residue 170 to
aspartic acid, modifying residue 171 to glutamine, modifying
residue 172 to methionine and/or modifying residue 173 to aspartic
acid. In a most preferred embodiment, the substitution comprises
modifying residues 170, 171 and 173 to aspartic acid, glutamine and
aspartic acid, respectively.
[0021] In another embodiment of the present invention, a method for
producing the protein of the invention having reduced allergenicity
is provided. Preferably, the mutant protein is prepared by
modifying a DNA encoding a precursor protein so that the modified
DNA encodes the mutant protein of the invention.
[0022] In yet another embodiment of the invention, DNA sequences
encoding the mutant protein, as well as expression vectors
containing such DNA sequences and host cells transformed with such
vectors are provided, which host cells are preferably capable of
expressing such DNA to produce the mutant protein of the invention
either intracellularly or extracellularly.
[0023] The mutant protein of the invention is useful in any
composition or process in which the precursor protein is generally
known to be useful. For example, where the protein is a protease,
the reduced allergenicity protease can be used as a component in
cleaning products such as laundry detergents and hard surface
cleansers, as an aid in the preparation of leather, in the
treatment of textiles such as wool and/or silk to reduce felting,
as a component in a personal care, cosmetic or face cream product,
and as a component in animal or pet feed to improve the nutritional
value of the feed. Similarly, where the protein is an amylase, the
reduce allergenicity amylase can be used for the liquefaction of
starch, as a component in a dishwashing detergent, for desizing of
textiles, in a laundry detergent or any other use for which amylase
is useful.
[0024] One advantage of the present invention is that by measuring
the proliferation of T-cells due to T-cell epitope recognition, it
is possible to identify peptides which contain epitopes responsible
for initially sensitizing an individual. That is, T-cell
proliferation due to T-cell epitope recognition results in
sensitization of an individual to that peptide or a protein which
contains it. Neutralization of such "sensitizing" T-cell epitopes
will inevitably result in a greater degree of safety for those who
handle or are otherwise exposed to the antigen containing the
epitope because they will not be initially sensitized, thus
preventing the production of Ig antibodies typical of an allergic
reaction upon subsequent exposure to the antigen.
[0025] An advantage of the present invention is the preparation of
proteins, including enzymes, which may be used with significantly
less danger of sensitization for the individuals exposed. Thus, for
example, the proteins of the invention may be more safely used in
cosmetics such as face creams, detergents such as laundry
detergents, hard surface cleaning compositions and-pre-wash
compositions or any other use of protein, including enzymes,
wherein human exposure is a necessary by-product.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGS. 1A, B1, B2 and B3 illustrate the DNA (SEQ ID:NO 1) and
amino acid (SEQ ID: NO 2) sequence for Bacillus amyloliquefaciens
subtilisin (BPN') and a partial restriction map of this gene.
[0027] FIG. 2 illustrates the conserved amino acid residues among
subtilisins from Bacillus amyloliquefaciens (SEQ ID:NO 3) and
Bacillus lentus (wild-type) (SEQ ID:NO 4).
[0028] FIGS. 3A and 3B illustrate an amino acid sequence alignment
of subtilisin type proteases from Bacillus amyloliquefaciens
(BPN'), Bacillus subtilis, Bacillus licheniformis (SEQ ID:NO 5) and
Bacillus lentus. The symbol * denotes the absence of specific amino
acid residues as compared to subtilisin BPN'.
[0029] FIG. 4 illustrates the additive T-cell response of 16
peripheral mononuclear blood samples to peptides corresponding to
the Bacillus lentus protease. Peptide E05 includes the region
comprising residues corresponding to 170-173 in protease from
Bacillus amyloliquefaciens.
[0030] FIG. 5 illustrates the additive T-cell response of 10
peripheral mononuclear blood samples to peptides corresponding to
the human subtilisin molecule. Peptides F10, F9, F8 and F7 all
contain the amino acid sequence DQMD corresponding to the region
comprising residues corresponding to 170-173 in protease from
Bacillus amyloliquefaciens in the sequence alignment of FIG. 3.
[0031] FIG. 6A and 6B/6C illustrate amino acid strings
corresponding to peptides derived from the sequence of Bacillus
lentus protease and a human subtilisin, respectively.
[0032] FIG. 7 illustrates the amino acid sequence of human
subtilisin (SEQ ID:NO 6).
[0033] FIG. 8 illustrates an amino acid sequence alignment of BPN'
(Bacillus ramyloliquefaciens) protease, SAVINASE (Bacillus lentus)
protease and human subtilisin (S2HSBT).
[0034] FIG. 9 illustrates the T-cell response to peptides derived
from Bacillus lentus protease in a sample taken from an individual
known to be hypersensitive to Bacillus lentus protease. Peptide E05
represents the region corresponding to 170-173 in protease from
Bacillus amyloliquefaciens.
[0035] FIG. 10 illustrates the T-cell response to various alanine
substitutions in the E05 Bacillus lentus protease peptide set in a
sample taken from an individual known to be hypersensitive to
Bacillus lentus protease.
DETAILED DESCRIPTION OF THE INVENTION
[0036] According to the present invention, a method for identifying
T-cell epitopes is provided. The present invention provides an
assay which identifies epitopes as follows: differentiated
dendritic cells are combined with naive human CD4+ and/or CD8+
T-cells and with a peptide of interest. More specifically, a method
is provided wherein a T-cell epitope is recognized comprising the
steps of: (a) obtaining from a single blood source a solution of
dendritic cells and a solution of naive CD4+ and/or CD8+ T-cells;
(b) promoting differentiation in said solution of dendritic cells;
(c) combining said solution of differentiated dendritic cells and
said naive CD4+ and/or CD8+ T-cells with a peptide of interest; (d)
measuring the proliferation of T-cells in said step (c).
[0037] The peptide of interest to be analyzed according to the
assay of the invention is derived from a protein or enzyme for
which reduced allergenicity is desirable or required. In the
practice of the invention, it is possible to identify with
precision the location of an epitope which can cause sensitization
in an individual or sampling of individuals. In a particularly
effective embodiment of the invention, a series of peptide
oligomers which correspond to all or part of the protein or enzyme
are prepared. For example, a peptide library is produced covering
the relevant portion or all of the protein. One particularly useful
manner of producing the peptides is to introduce overlap into the
peptide library, for example, producing a first peptide corresponds
to amino acid sequence 1-10 of the subject protein, a second
peptide corresponds to amino acid sequence 4-14 of the subject
protein, a third peptide corresponds to amino acid sequence 7-17 of
the subject protein, a fourth peptide corresponds to amino acid
sequence 10-20 of the subject protein etc. . . . until
representative peptides corresponding to the entire molecule are
created. By analyzing each of the peptides individually in the
assay provided herein, it is possible to precisely identify the
location of epitopes recognized by T-cells. In the example above,
the reaction of one specific peptide to a greater extent than it's
neighbors will facilitate identification of the epitope anchor
region to within three amino acids. After determining the location
of these epitopes, it is possible to alter the amino acids within
each epitope until the peptide produces a less significant T-cell
response.
[0038] "Antigen presenting cell" as used herein means a cell of the
immune system which present antigen on their surface which is
recognizable by receptors on the surface of T-cells. Examples of
antigen presenting cells are dendritic cells, interdigitating
cells, activated B-cells and macrophages.
[0039] "T-cell proliferation" as used herein means the number of
T-cells produced during the incubation of T-cells with the antigen
presenting cells, with or without antigen.
[0040] "Baseline T-cell proliferation" as used herein means T-cell
proliferation which is normally seen in an individual in response
to exposure to antigen presenting cells in the absence of peptide
or protein antigen. For the purposes herein, the baseline T-cell
proliferation level was determined on a per sample basis for each
individual as the proliferation of T-cells in response to antigen
presenting cells in the absence of antigen.
[0041] "T-cell epitope" means a feature of a peptide or protein
which is recognized by a T-cell receptor in the initiation of an
immunologic response to the peptide comprising that antigen.
Recognition of a T-cell epitope by a T-cell is generally believed
to be via a mechanism wherein T-cells recognize peptide fragments
of antigens which are bound to class I or class II major
histocompatability (MHC) molecules expressed on antigen-presenting
cells (see e.g., Moeller, G. ed., Antigenic Requirements for
Activation of MHC-Restricted Responses, Immunological Review, Vol.
98, p. 187 (Copenhagen; Munksgaard) (1987).
[0042] The epitopes determined according to the assay provided
herein are then modified to reduce the allergenic potential of the
protein of interest. In a preferred embodiment, the epitope to be
modified produces a level of T-cell proliferation of greater than
three times the baseline T-cell proliferation in a sample. When
modified, the epitope produces less than three times the baseline
proliferation, preferably less than two times the baseline
proliferation and most preferably less than or substantially equal
to the baseline proliferation in a sample..
[0043] Preferably, the epitope is modified in one of the following
ways: (a) the amino acid sequence of the epitope is substituted
with an analogous sequence from a human homolog to the protein of
interest; (b) the amino acid sequence of the epitope is substituted
with an analogous sequence from a non-human homolog to the protein
of interest, which analogous sequence produces a lesser allergenic
response due to T-cell epitope recognition than that of the protein
of interest; (c) the amino acid sequence of the epitope is
substituted with a sequence which substantially mimics the major
tertiary structure attributes of the epitope, but which produces a
lesser allergenic response due to T-cell epitope recognition than
that of the protein of interest; or (d) with any sequence which
produces lesser allergenic response due to T-cell epitope
recognition than that of the protein of interest.
[0044] "Sample" as used herein comprises mononuclear cells which
are naive, i.e., not sensitized, to the antigen in question.
[0045] "Homolog" as used herein means a protein or enzyme which has
similar catalytic action, structure and/or use as the protein of
interest. It is desirable to find a homolog that has a tertiary
and/or primary structure similar to the protein of interest as
replacement of the epitope in the protein of interest with an
analogous segment from the homolog will reduce the disruptiveness
of the change. Thus, closely homologous enzymes will provide the
most desirable source of epitope substitutions. Alternatively, if
possible, it is advantageous to look to human analogs for a given
protein. For example, substituting a specific epitope in a
bacterial subtilisin with a sequence from a human analog to
subtilisin (i.e., human subtilisin) should result in less
allergenicity in the bacterial protein.
[0046] An "analogous" sequence may be determined by ensuring that
the replacement amino acids show a similar function, the tertiary
structure and/or conserved residues to the amino acids in the
protein of interest at or near the epitope. Thus, where the epitope
region contains, for example, an alpha-helix or a beta-sheet
structure, the replacement amino acids should maintain that
specific structure.
[0047] While the present invention extends to all proteins for
which it is desired to reduce allergenicity, for the sake of
simplicity, the following will describe a particularly preferred
embodiment of the invention, the modification of protease.
Proteases are carbonyl hydrolases which generally act to cleave
peptide bonds of proteins or peptides. As used herein, "protease"
means a naturally-occurring protease or a recombinant protease.
Naturally-occurring proteases include .alpha.-aminoacylpeptide
hydrolase, peptidylamino acid hydrolase, acylamino hydrolase,
serine carboxypeptidase, metallocarboxypeptidase, thiol proteinase,
carboxylproteinase and metalloproteinase. Serine, metallo, thiol
and acid proteases are included, as well as endo and
exo-proteases.
[0048] Subtilisins are bacterial or fungal proteases which
generally act to cleave peptide bonds of proteins or peptides. As
used herein, "subtilisin" means a naturally-occurring subtilisin or
a recombinant subtilisin. A series of naturally-occurring
subtilisins is known to be produced and often secreted by various
microbial species. Amino acid sequences of the members of this
series are not entirely homologous. However, the subtilisins in
this series exhibit the same or similar type of proteolytic
activity. This class of serine proteases shares a common amino acid
sequence defining a catalytic triad which distinguishes them from
the chymotrypsin related, class of serine proteases. The
subtilisins and chymotrypsin related serine proteases both have a
catalytic triad comprising aspartate, histidine and serine. In the
subtilisin related proteases the relative order of these amino
acids, reading from the amino to carboxy terminus, is
aspartate-histidine-serine. In the chymotrypsin related proteases,
the relative order, however, is histidine-aspartate-serine. Thus,
subtilisin herein refers to a serine protease having the catalytic
triad of subtilisin related proteases. Examples include but are not
limited to the subtilisins identified in FIG. 3 herein. Generally
and for purposes of the present invention, numbering of the amino
acids in proteases corresponds to the numbers assigned to the
mature Bacillus amyloliquefaciens subtilisin sequence presented in
FIG. 1.
[0049] "Recombinant subtilisin" or "recombinant protease" refer to
a subtilisin or protease in which the DNA sequence encoding the
subtilisin or protease is modified to produce a variant (or mutant)
DNA sequence which encodes the substitution, deletion or insertion
of one or more amino acids in the naturally-occurring amino acid
sequence. Suitable methods to produce such modification, and which
may be combined with those disclosed herein, include those
disclosed in U.S. Pat. No. 4,760,025 (RE 34,606), U.S. Pat. No.
5,204,015 and U.S. Pat. No. 5,185,258.
[0050] "Non-human subtilisins" and the DNA encoding them may be
obtained from many procaryotic and eucaryotic organisms. Suitable
examples of procaryotic organisms include gram negative organisms
such as E. coli or Pseudomonas and gram positive bacteria such as
Micrococcus or Bacillus. Examples of eucaryotic organisms from
which subtilisin and their genes may be obtained include yeast such
as Saccharomyces cerevisiae, fungi such as Aspergillus sp.
[0051] "Human subtilisin" means proteins of human origin which have
subtilisin type catalytic activity, e.g., the kexin family of human
derived proteases. An example of such a protein is represented by
the sequence in FIG. 7. Additionally, derivatives or homologs of
human subtilisin, including those from non-human sources such as
mouse or rabbit, which retain the essential ability to hydrolyze
peptide bonds and have at least 50%, preferably at least 65% and
most preferably at least 80% homology to the protein of FIG. 7 are
considered human subtilisins for the purpose of the invention.
[0052] A "protease variant" has an amino acid sequence which is
derived from the amino acid sequence of a "precursor protease". The
precursor proteases include naturally-occurring proteases and
recombinant proteases. The amino acid sequence of the protease
variant is "derived" from the precursor protease amino acid
sequence by the substitution, deletion or insertion of one or more
amino acids of the precursor amino acid sequence. Such modification
is of the "precursor DNA sequence" which encodes the amino acid
sequence of the precursor protease rather than manipulation of the
precursor protease enzyme per se. Suitable methods for such
manipulation of the precursor DNA sequence include
methods-disclosed herein, as well as methods known to those skilled
in the art (see, for example, EP 0 328299, WO89/06279 and the U.S.
patents and applications already referenced herein).
[0053] The amino acid position numbers used herein refer to those
assigned to the mature Bacillus amyloliquefaciens subtilisin
sequence presented in FIG. 1. The invention, however, is not
limited to the mutation of this particular subtilisin but extends
to precursor proteases containing amino acid residues at positions
which are "equivalent" to the particular identified residues in
Bacillus amyloliquefaciens subtilisin. In a preferred embodiment of
the present invention, the precursor protease is Bacillus lentus
subtilisin and the substitutions, deletions or insertions are made
at the equivalent amino acid residue in B. lentus corresponding to
those listed above.
[0054] A residue (amino acid) of a precursor protease is equivalent
to a residue of Bacillus amyloliquefaciens subtilisin if it is
either homologous (i.e., corresponding in position in either
primary or tertiary structure) or analogous to a specific residue
or portion of that residue in Bacillus amyloliquefaciens subtilisin
(i.e., having the same or similar functional capacity to combine,
react, or interact chemically).
[0055] In order to establish homology to primary structure, the
amino acid sequence of a precursor protease is directly compared to
the Bacillus amyloliquefaciens subtilisin primary sequence and
particularly to a set of residues known to be invariant in
subtilisins for which the sequence is known. For example, FIG.
2,herein shows the conserved residues as between B.
amyloliquefaciens subtilisin and B. lentus subtilisin. After
aligning the conserved residues, allowing for necessary insertions
and deletions in order to maintain alignment (i.e., avoiding the
elimination of conserved residues through arbitrary deletion and
insertion), the residues equivalent to particular amino acids in
the primary sequence of Bacillus amyloliquefaciens subtilisin are
defined. Alignment of conserved residues preferably should conserve
100% of such residues. However, alignment of greater than 75% or as
little as 50% of conserved residues is also adequate to define
equivalent residues. Conservation of the catalytic triad,
Asp32/His64/Ser221 should be maintained.
[0056] For example, the amino acid sequence of subtilisin from
Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus
licheniformis (carlsbergensis) and Bacillus lentus can be aligned
to provide the maximum amount of homology between amino acid
sequences. A comparison of these sequences shows that there are a
number of conserved residues contained in each sequence. The
conserved residues as between BPN' and B. lentus are identified in
FIG. 2.
[0057] These conserved residues, thus, may be used to define the
corresponding equivalent amino acid residues of Bacillus
amyloliquefaciens subtilisin in other subtilisins such as
subtilisin from Bacillus lentus (PCT Publication No. WO89/06279
published Jul. 13, 1989), the preferred protease precursor enzyme
herein, or the subtilisin referred to as PB92 (EP 0 328 299), which
is highly homologous to the preferred Bacillus lentus subtilisin.
The amino acid sequences of certain of these subtilisins are
aligned in FIGS. 3A and 3B with the sequence of Bacillus
amyloliquefaciens subtilisin to produce the maximum homology of
conserved residues. As can be seen, there are a number of deletions
in the sequence of Bacillus lentus as compared to Bacillus
amyloliquefaciens subtilisin. Thus, for example, the equivalent
amino acid for Val165 in Bacillus amyloliquefaciens subtilisin in
the other subtilisins is isoleucine for B. lentus and B.
licheniformis.
[0058] Thus, for example, the amino acid at position +170 is lysine
(K) in both B. amyloliquefaciens and B. licheniformis subtilisins
and arginine (R) in Savinase. In one embodiment of the protease
variants of the invention, however, the amino acid equivalent to
+170 in Bacillus amyloliquefaciens subtilisin is substituted with
aspartic acid (D). The abbreviations and one letter codes for all
amino acids in the present invention conform to the Patentin User
Manual (GenBank, Mountain View, Calif.) 1990, p.101.
[0059] "Equivalent residues" may also be defined by determining
homology at the level of tertiary structure for a precursor
protease whose tertiary structure has been determined by x-ray
crystallography. Equivalent residues are defined as those for which
the atomic coordinates of two or more of the main chain atoms of a
particular amino acid residue of the precursor protease and
Bacillus amyloliquefaciens subtilisin (N on N, CA on CA, C on C and
O on O) are within 0.13 nm and preferably 0.1 nm after alignment.
Alignment is achieved after the best model has been oriented and
positioned to give the maximum overlap of atomic coordinates of
non-hydrogen protein atoms of the protease in question to the
Bacillus amyloliquefaciens subtilisin. The best model is the
crystallographic model giving the lowest R factor for experimental
diffraction data at the highest resolution available. 1 R factor =
h Fo ( h ) - Fc ( h ) h Fo ( h )
[0060] Equivalent residues which are functionally analogous to a
specific residue of Bacillus amyloliquefaciens subtilisin are
defined as those amino acids of the precursor protease which may
adopt a conformation such that they either alter, modify or
contribute to protein structure, substrate binding or catalysis in
a manner defined and attributed to a specific residue of the
Bacillus amyloliquefaciens subtilisin. Further, they are those
residues of the precursor protease (for which a tertiary structure
has been obtained by x-ray crystallography) which occupy an
analogous position to the extent that, although the main chain
atoms of the given residue may not satisfy the criteria of
equivalence on the basis of occupying a homologous position, the
atomic coordinates of at least two of the side chain atoms of the
residue lie with 0.1 3 nm of the corresponding side chain atoms of
Bacillus amyloliquefaciens subtilisin. The coordinates of the three
dimensional structure of Bacillus amyloliquefaciens subtilisin are
set forth in EPO Publication No. 0 251 446 (equivalent to U.S. Pat.
No. 5,182,204, the disclosure of which is incorporated herein by
reference) and can be used as outlined above to determine
equivalent residues on the level of tertiary structure.
[0061] Some of the residues identified for substitution, insertion
or deletion are conserved residues whereas others are not. In the
case of residues which are not conserved, the replacement of one or
more amino acids is limited to substitutions which produce a
variant which has an amino acid sequence that does not correspond
to one found in nature. In the case of conserved residues, such
replacements should not result in a naturally-occurring sequence.
The protease variants of the present invention include the mature
forms of protease variants, as well as the pro- and prepro-forms of
such protease variants. The prepro-forms are the preferred
construction since this facilitates the expression, secretion and
maturation of the protease variants.
[0062] "Prosequence" refers to a sequence of amino acids bound to
the N-terminal portion of the mature form of a protease which when
removed results in the appearance of the "mature" form of the
protease. Many proteolytic enzymes are found in nature as
translational proenzyme products and, in the absence of
post-translational processing, are expressed in this fashion. A
preferred prosequence for producing protease variants is the
putative prosequence of Bacillus amyloliquefaciens subtilisin,
although other protease prosequences may be used.
[0063] A "signal sequence" or "presequence" refers to any sequence
of amino acids bound to the N-terminal portion of a protease or to
the N-terminal portion of a proprotease which may participate in
the secretion of the mature or pro forms of the protease. This
definition of signal sequence is a functional one, meant to include
all those amino acid sequences encoded by the N-terminal portion of
the protease gene which participate in the effectuation of the
secretion of protease under native conditions. The present
invention utilizes such sequences to effect the secretion of the
protease variants as defined herein. One possible signal sequence
comprises the first seven amino acid residues of the signal
sequence from Bacillus subtilis subtilisin fused to the remainder
of the signal sequence of the subtilisin from Bacillus lentus (ATCC
21536).
[0064] A "prepro" form of a protease variant consists of the mature
form of the protease having a prosequence operably linked to the
amino terminus of the protease and a "pre" or "signal" sequence
operably linked to the amino terminus of the prosequence.
[0065] "Expression vector" refers to a DNA construct containing a
DNA sequence which is operably linked to a suitable control
sequence capable of effecting the expression of said DNA in a
suitable host. Such control sequences include a promoter to effect
transcription, an optional operator sequence to control such
transcription, a sequence encoding suitable mRNA ribosome binding
sites-and sequences which control termination of transcription and
translation. The vector may be a plasmid, a phage particle, or
simply a potential genomic insert. Once transformed into a suitable
host, the vector may replicate and function independently of the
host genome, or may, in some instances, integrate into the genome
itself. In the present specification, "plasmid" and "vector" are
sometimes used interchangeably as the plasmid is the most commonly
used form of vector at present. However, the invention is intended
to include such other forms of expression vectors which serve
equivalent functions and which are, or become, known in the
art.
[0066] The "host cells" used in the present invention generally are
procaryotic or eucaryotic hosts which preferably have been
manipulated by the methods disclosed in U.S. Pat. No. 4,760,025 (RE
34,606) to render them incapable of secreting enzymatically active
endoprotease. A preferred host cell for expressing protease is the
Bacillus strain BG2036 which is deficient in enzymatically active
neutral protease and alkaline protease (subtilisin). The
construction of strain BG2036 is described in detail in U.S. Pat.
No. 5,264,366. Other host cells for expressing protease include
Bacillus subtilis I168 (also described in U.S. Pat. No. 4,760,025
(RE 34,606) and U.S. Pat. No. 5,264,366, the disclosure of which
are incorporated herein by reference), as well as any suitable
Bacillus strain such as B. licheniformis, B. lentus, etc.
[0067] Host cells are transformed or transfected with vectors
constructed using recombinant DNA techniques. Such transformed host
cells are capable of either replicating vectors encoding the
protease variants or expressing the desired protease variant. In
the case of vectors which encode the pre- or prepro-form of the
protease variant, such variants, when expressed, are typically
secreted from the host cell into the host cell medium.
[0068] "Operably linked", when describing the relationship between
two DNA regions, simply means that they are functionally related to
each other. For example, a presequence is operably linked to a
peptide if it functions as a signal sequence, participating in the
secretion of the mature form of the protein most probably involving
cleavage of the signal sequence. A promoter is operably linked to a
coding sequence if it controls the transcription of the sequence; a
ribosome binding site is operably linked to a coding sequence if it
is positioned so as to permit translation.
[0069] The genes encoding the naturally-occurring precursor
protease may be obtained in accord with the general methods known
to those skilled in the art. The methods generally comprise
synthesizing labeled probes having putative sequences encoding
regions of the protease of interest, preparing genomic libraries
from organisms expressing the protease, and screening the libraries
for the gene of interest by hybridization to the probes. Positively
hybridizing clones are then mapped and sequenced.
[0070] The cloned protease is then used to transform a host cell in
order to express the protease. The protease gene is then ligated
into a high copy number plasmid. This plasmid replicates in hosts
in the sense that it contains the well-known elements necessary for
plasmid replication: a promoter operably linked to the gene in
question (which may be supplied as the gene's own homologous
promoter if it is recognized, i.e., transcribed, by the host),
a-transcription termination and polyadenylation region (necessary
for stability of the mRNA transcribed by the host from the protease
gene in certain eucaryotic host cells) which is exogenous or is
supplied by the endogenous terminator region of the protease gene
and, desirably, a selection gene such as an antibiotic resistance
gene that enables continuous cultural maintenance of
plasmid-infected host cells by growth in antibiotic-containing
media. High copy number plasmids also contain an origin of
replication for the host, thereby enabling large numbers of
plasmids to be generated in the cytoplasm without chromosomal
limitations. However, it is within the scope herein to integrate
multiple copies of the protease gene into host genome. This is
facilitated by procaryotic and eucaryotic organisms which are
particularly susceptible to homologous recombination.
[0071] In one embodiment, the gene can be a natural gene such as
that from B lentus or B. amyloliquefaciens. Alternatively, a
synthetic gene encoding a naturally-occurring or mutant precursor
protease may be produced. In such an approach, the DNA and/or amino
acid sequence of the precursor protease is determined. Multiple,
overlapping synthetic single-stranded DNA fragments are thereafter
synthesized, which upon hybridization and ligation produce a
synthetic DNA encoding the precursor protease. An example of
synthetic gene construction is set forth in Example 3 of U.S. Pat.
No. 5,204,015, the disclosure of which is incorporated herein by
reference.
[0072] Once the naturally-occurring or synthetic precursor protease
gene has been cloned, a number of modifications are undertaken to
enhance the use of the gene beyond synthesis of the
naturally-occurring precursor protease. Such modifications include
the production of recombinant proteases as disclosed in U.S. Pat.
No. 4,760,025 (RE 34,606) and EPO Publication No. 0 251 446 and the
production of protease variants described herein.
[0073] The following cassette mutagenesis method may be used to
facilitate the construction of the protease variants of the present
invention, although other methods may be used. First, the
naturally-occurring gene encoding the protease is obtained and
sequenced in whole of in part. Then the sequence is scanned for a
point at which it is desired to make a mutation (deletion,
insertion or substitution) of one or more amino acids in the
encoded enzyme. The sequences flanking this point are evaluated for
the presence of restriction sites for replacing a short segment of
the gene with an oligonucleotide pool which when expressed will
encode various mutants. Such restriction sites are preferably
unique sites within the protease gene so as to facilitate the
replacement of the gene segment. However, any convenient
restriction site which is not overly redundant in the protease gene
may be used, provided the gene fragments generated by restriction
digestion can be reassembled in proper sequence. If restriction
sites are not present at locations within a convenient distance
from the selected point (from 10 to 15 nucleotides), such sites are
generated by substituting nucleotides in the gene in such a fashion
that neither the reading frame nor the amino acids encoded are
changed in the final construction. Mutation of the gene in order to
change its sequence to conform to the desired sequence is
accomplished by M13 primer extension in accord with generally known
methods. The task of locating suitable flanking regions and
evaluating the needed changes to arrive at two convenient
restriction site sequences is made routine by the redundancy of the
genetic code, a restriction enzyme map of the gene and the large
number of different restriction enzymes. Note that if a convenient
flanking restriction site is available, the above method need be
used only in connection with the flanking region which does not
contain a site.
[0074] Once the naturally-occurring DNA or synthetic DNA is cloned,
the restriction sites flanking the positions to be mutated are
digested with the cognate restriction enzymes and a plurality of
end termini-complementary oligonucleotide cassettes are ligated
into the gene. The mutagenesis is simplified by this method because
all of the oligonucleotides can be synthesized so as to have the
same restriction sites, and no synthetic linkers are necessary to
create the restriction sites.
[0075] In one aspect of the invention, the objective is to secure a
variant protease having altered allergenic potential as compared to
the precursor protease, since decreasing such potential enables
safer use of the enzyme. While the instant invention is useful to
lower allergenic potential, the mutations specified herein may be
utilized in combination with mutations known in the art to result
altered thermal stability and/or altered substrate specificity,
modified activity or altered alkaline stability as compared to the
precursor.
[0076] Accordingly, the present invention is directed to altering
the capability of the T-cell epitope which includes residue
positions 170-173 in Bacillus lentus to induce T-cell
proliferation. One particularly preferred embodiment of the
invention comprises making modification to either one or all of
R170D, Y171Q and/or N173D. Similarly, as discussed in detail above,
it is believed that the modification of the corresponding residues
in any protease will result in a the neutralization of a key T-cell
epitope in that protease. Thus, in combination with the presently
disclosed mutations in the region corresponding to amino acid
residues 170-173, substitutions at positions corresponding to
N76D/S103A/V104I/G159D optionally in combination with one or more
substitutions selected from the group consisting of positions
corresponding to V68A, T213R, A232V, Q236H, Q245R, and T260A of
Bacillus amyloliquefaciens subtilisin may be used, in addition to
decreasing the allergenic potential of the variant protease of the
invention, to modulate overall stability and/or proteolytic
activity of the enzyme. Similarly, the substitutions provided
herein may be combined with mutation at the Asparagine (N) in
Bacillus lentus subtilisin at equivalent position +76 to Aspartate
(D) in combination with the mutations S103AN1041/G159D and
optionally in combination with one or more substitutions selected
from the group consisting of positions corresponding to V68A,
T213R, A232V, Q236H, Q245R, and T260A of Bacillus amyloliquefaciens
subtilisin, to produce enhanced stability and/or enhanced activity
of the resulting mutant enzyme.
[0077] The most preferred embodiments of the invention include the
following specific combinations of substituted residues
corresponding to positions:
N76D/S103A/V104I/G159D/K170D/Y171Q/S173D;
V68A/N76D/S103AN104I/G159D/K170D/Y171Q/S173D/Q236H;
V68A/N76D/S103AN/104I/G159D/K170D/Y171 Q/S173D/Q236H/Q245R;
V68A/N76D/S103A/V104I/G159D/K170D/Y171 Q/S173D/A232V/Q236H/Q245R;
and V68A/N76D//S103AN104I/G159D/K170D/Y171
Q/S173D/T213R/A232V/Q236H/Q245R/T2- 60A of Bacillus
amyloliquefaciens subtilisin. These substitutions are preferably
made in Bacillus lentus (recombinant or native-type) subtilisin,
although the substitutions may be made in any Bacillus
protease.
[0078] Based on the screening results obtained with the variant
proteases, the noted mutations noted above in Bacillus
amyloliquefaciens subtilisin are important to the proteolytic
activity, performance and/or stability of these enzymes and the
cleaning or wash performance of such variant enzymes.
[0079] Many of the protease variants of the invention are useful in
formulating various detergent compositions. A number of known
compounds are suitable surfactants useful in compositions
comprising the protease mutants of the invention. These include
nonionic, anionic, cationic, anionic or zwitterionic detergents, as
disclosed in U.S. Pat. No. 4,404,128 to Barry J. Anderson and U.S.
Pat. No. 4,261,868 to Jiri Flora, et al. A suitable detergent
formulation is that described in Example 7 of U.S. Pat. No.
5,204,015 (previously incorporated by reference). The art is
familiar with the different formulations which can be used as
cleaning compositions. In addition to typical cleaning
compositions, it is readily understood that the protease variants
of the present invention may be used for any purpose that native or
wild-type proteases are used. Thus, these variants can be used, for
example, in bar or liquid soap applications, dishcare formulations,
contact lens cleaning solutions or products, peptide hydrolysis,
waste treatment, textile applications, as fusion-cleavage enzymes
in protein production, etc. The variants of the present invention
may comprise, in addition to decreased allergenicity, enhanced
performance in a detergent composition (as compared to the
precursor). As used herein, enhanced performance in a detergent is
defined as increasing cleaning of certain enzyme sensitive stains
such as grass or blood, as determined by usual evaluation after a
standard wash cycle.
[0080] Proteases of the invention can be formulated into known
powdered and liquid detergents having pH between 6.5 and 12.0 at
levels of about 0.01 to about 5% (preferably 0.1% to 0.5%) by
weight. These detergent cleaning compositions can also include
other enzymes such as known proteases, amylases, cellulases,
lipases or endoglycosidases, as well as builders and
stabilizers.
[0081] The addition of proteases of the invention to conventional
cleaning compositions does not create any special use limitation.
In other words, any temperature and pH suitable for the detergent
is also suitable for the present compositions as long as the pH is
within the above range, and the temperature is below the described
protease's denaturing temperature. In-addition, proteases of the
invention can be used in a cleaning composition without detergents,
again either alone or in combination with builders and
stabilizers.
[0082] The variant proteases of the present invention can be
included in animal feed such as part of animal feed additives as
described in, for example, U.S. Pat. No. 5,612,055; U.S. Pat. No.
5,314,692; and U.S. Pat. No. 5,147,642.
[0083] One aspect of the invention is a composition for the
treatment of a textile that includes variant proteases of the
present invention. The composition can be used to treat for example
silk or wool as described in publications such as RD 216,034; EP
134,267; U.S. Pat. No. 4,533,359; and EP 344,259.
[0084] The following is presented by way of example and is not to
be construed as a limitation to the scope of the claims.
[0085] The variants can be screened for proteolytic activity
according to methods well known in the art. Preferred protease
variants include multiple substitutions at positions corresponding
to: N76D/S103A/V104I/G159D/K170D/Y171 Q/S173D;
V68A/N76D/S103A/V104I/G159D/K1- 70D/Y171Q/S173D/Q236H;
V68A/N76D/S103A/V104I/G159D/K170D/Y171Q/S173D/Q236H- /Q245R;
V68A/N76D/S103A/V104I/G159D/K170D/Y171Q/S173D/A232V/Q236H/Q245R;
and
V68A/N76D/S103A/V104I/G159D/K170D/Y171Q/S173D/T213R/A232V/Q236H/Q245
R/T260A of Bacillus amyloliquefaciens subtilisin.
[0086] All publications and patents referenced herein are hereby
incorporated by reference in their entirety.
EXAMPLES
Example 1
Assay for the Identification of Peptide T-Cell Epitopes Using Naive
Human T-Cells
[0087] Fresh human peripheral blood cells were collected from
"naive" humans, i.e., persons not known to be exposed to or
sensitized to Bacillus lentus protease, for determination of
antigenic epitopes in protease from Bacillus lentus and human
subtilisin. Naive humans is intended to mean that the individual is
not known to have been exposed to or developed a reaction to
protease in the past. Peripheral mononuclear blood cells (stored at
room temperature, no older than 24 hours) were prepared for use as
follows: Approximately 30 mls of a solution of buffy coat
preparation from one unit of whole blood was brought to 50 ml with
Dulbecco's phosphate buffered solution (DPBS) and split into two
tubes. The samples were underlaid with 12.5 ml of room temperature
lymphoprep density separation media (Nycomed density 1.077 g/ml).
The tubes were centrifuged for thirty minutes at 600 G. The
interface of the two phases was collected, pooled and washed in
DPBS. The cell density of the resultant solution was measured by
hemocytometer. Viability was measured by trypan blue exclusion.
[0088] From the resulting solution, a differentiated dendritic cell
culture was prepared from the peripheral blood mononuclear cell
sample having a density of 10.sup.8 cells per 75 ml culture flask
in a solution as follows:
[0089] (1) 50 ml of serum free AIM V media (Gibco) was supplemented
with a 1:100 dilution beta-mercaptoethanol (Gibco). The flasks were
laid flat for two hours at 37.degree. C. in 5% CO.sub.2 to allow
adherence of monocytes to the flask wall.
[0090] (2) Differentiation of the monocyte cells to dendritic cells
was as follows: nonadherent cells were removed and the resultant
adherent cells (monocytes) combined with 30 ml of AIM V, 800
units/ml of GM-CSF (Endogen) and 500 units/ml of IL-4 (Endogen);
the resulting mixture was cultured for 5 days under conditions at
37.degree. C. in 5% CO.sub.2. After five days, the cytokine
TNF.alpha. (Endogen) was added to 0.2 units/ml, and the cytokine
IL-1.alpha. (Endogen) was added to a final concentration of 50
units/ml and the mixture incubated at 37.degree. C. in 5% CO.sub.2
for two more days.
[0091] (3) On the seventh day, Mitomycin C was added to a
concentration of 50 microgram/ml was added to stop growth of the
now differentiated dendritic cell culture. The solution was
incubated for 60 minutes at 37.degree. C. in 5% CO.sub.2. Dendritic
cells were collected by gently scraping the adherent cells off the
bottom of the flask with a cell scraper. Adherent and non-adherent
cells were then centrifuged at 600 G for 5 minutes, washed in DPBS
and counted.
[0092] (4) The prepared dendritic cells were placed into a 96 well
round bottom array at 2.times.10.sup.4/well in 100 microliter total
volume of AIM V media.
[0093] CD4+ T cells were prepared from frozen aliquots of the
peripheral blood cell samples used to prepare the dendritic cells
using the human CD4+ Cellect Kit (Biotex) as per the manufacturers
instructions with the following modifications: the aliquots were
thawed and washed such that approximately 10.sup.8 cells will be
applied per Cellect column; the cells were resuspended in 4 ml DPBS
and 1 ml of the Cell reagent from the Cellect Kit, the solution
maintained at room temperature for 20 minutes. The resultant
solution was centrifuged for five minutes at 600G at room
temperature and the pellet resuspended in 2 ml of DPBS and applied
to the Cellect columns. The effluent from the columns was collected
in 2% human serum in DPBS. The resultant CD4+ cell solution was
centrifuged, resuspended in AIMV media and the density counted.
[0094] The CD4+ T-cell suspension was resuspended to a count of
2.times.10.sup.6/ml in AIM V media to facilitate efficient
manipulation of the 96 well plate.
[0095] Peptide antigen is prepared from a 1M stock solution in DMSO
by dilution in AIM V media at a 1:10 ratio. 10 microliters of the
stock solution is placed in each well of the 96 well plate
containing the differentiated dendritic cells. 100 microliter of
the diluted CD4+ T-cell solution as prepared above is further added
to each well. Useful controls include diluted DMSO blanks, and
tetanus toxoid positive controls.
[0096] The final concentrations in each well, at 210 microliter
total volume are as follows:
[0097] 2.times.10.sup.4 CD4+
[0098] 2.times.10.sup.5 dendtritic cells (R:S of 10:1)
[0099] 5 mM peptide
Example 2
Identification of T-Cell Epitopes in Protease from Bacillus lentus
and Human subtilisin
[0100] Peptides for use in the assay described in Example 1 were
prepared based on the Bacillus lentus and human subtilisin amino
acid sequence. Peptide antigens were designed as follows. From the
full length amino acid sequence of either human subtilisin or
Bacillus lentus protease provided in FIG. 1, 15 mers were
synthetically prepared, each 15 mer overlapping with the previous
and the subsequent 15 mer except for three residues.
[0101] Peptides used correspond to amino acid residue strings in
Bacillus lentus as provided in FIG. 8, and peptides correspond to
amino acid residues in human subtilisin as provided in FIG. 7. The
peptides used corresponding to the proteases is provided in FIG. 6.
All tests were performed at least in duplicate. All tests reported
displayed robust positive control responses to the antigen tetanus
toxoid. Responses were averaged within each experiment, then
normalized to the baseline response. A positive event was recorded
if the response was at least 3 times the baseline response.
[0102] The immunogenic response (i.e., T-cell proliferation) to the
prepared peptides from human subtilisin and Bacillus lentus was
tallied and is provided in FIGS. 4 and 5, respectively. T-cell
proliferation was measured by the incorporated tritium method. The
results shown in FIGS. 4 and 5 as a comparison of the immunogenic
additive response in 10 individuals (FIG. 4) and 16 individuals
(FIG. 5) to the various peptides. Response is indicated as the
added response wherein 1.0 equals a baseline response for each
sample. Thus, in FIG. 4, a reading of 10.0 or less is the baseline
response and in FIG. 5 a reading of 16.0 or less the baseline
response.
[0103] As indicated in FIGS. 4 and 5, the immunogenic response of
the naive blood samples from unsensitized individuals showed a
marked allergenic response at the peptide fragment from Bacillus
lentus corresponding to residues 170-173 of Bacillus
amyloliquefaciens protease. As expected, the corresponding fragment
in human subtilisin evokes merely baseline response.
[0104] FIG. 9 shows the T-cell response to peptides derived from
Bacillus lentus protease in a sample taken from an individual known
to be hypersensitive to Bacillus lentus protease. Peptide E05
represents the region corresponding to 170-173 in protease from
Bacillus amyloliquefaciens. As shown in FIG. 9, the hypersensitive
individual was highly responsive to the T-cell epitope represented
by the peptide E05. This result confirms that, by practicing the
assay according to the invention, it is possible to predict the
major epitopes identified by the T-c%Ils of a hypersensitive
individual.
[0105] FIG. 10 shows the T-cell response to various alanine
substitutions in the E05 peptide derived from Bacillus lentus
protease in a sample taken from an individual known to be
hypersensitive to Bacillus lentus protease. Alanine substitutions
were used as substitutions for the purpose of determining the role
of any specific residue within the epitope. The legend of FIG. 10
refers to the position of the peptide in which an alanine was
substituted, i.e., in peptide E06 (sequence GSISYPARYANAMAV), G to
A=2, S to A=3,1 to A=4, S to A=5, Y to A=6, P to A=7, R to A=8, Y
to A=9, N to A=10, M to A=11 and V to A=12. As indicated in FIG.
10, substitution of either of the residues R170A, Y171A and/or
N173A in protease from Bacillus lentus results in dramatically
reduced response in the hypersensitive individual's blood
sample.
[0106] From these results, it is apparent that the residues 170,
171 and 173 are critical for T-cell response within this peptide.
Accordingly, it is further apparent that these residues are largely
responsible for the initiation of allergic reaction within the
protease from Bacillus lentus.
Sequence CWU 1
1
211 1 1495 DNA Bacillus amyloliquefaciens mat_peptide (417)..(1495)
CDS (96)..(1244) misc_feature (96)..(98) In one embodiment, the
"nnn" at positions 96 through 98 represents "atg", which in a
preferred embodiment codes for methionine. The "nnn" may also
represent "gtg", which codes for valine. "Xaa" represents either of
these amino acids. 1 ggtctactaa aatattattc catactatac aattaataca
cagaataatc tgtctattgg 60 ttattctgca aatgaaaaaa aggagaggat aaaga nnn
aga ggc aaa aaa gta 113 Xaa Arg Gly Lys Lys Val -105 tgg atc agt
ttg ctg ttt gct tta gcg tta atc ttt acg atg gcg ttc 161 Trp Ile Ser
Leu Leu Phe Ala Leu Ala Leu Ile Phe Thr Met Ala Phe -100 -95 -90
ggc agc aca tcc tct gcc cag gcg gca ggg aaa tca aac ggg gaa aag 209
Gly Ser Thr Ser Ser Ala Gln Ala Ala Gly Lys Ser Asn Gly Glu Lys -85
-80 -75 -70 aaa tat att gtc ggg ttt aaa cag aca atg agc acg atg agc
gcc gct 257 Lys Tyr Ile Val Gly Phe Lys Gln Thr Met Ser Thr Met Ser
Ala Ala -65 -60 -55 aag aag aaa gat gtc att tct gaa aaa ggc ggg aaa
gtg caa aag caa 305 Lys Lys Lys Asp Val Ile Ser Glu Lys Gly Gly Lys
Val Gln Lys Gln -50 -45 -40 ttc aaa tat gta gac gca gct tca gct aca
tta aac gaa aaa gct gta 353 Phe Lys Tyr Val Asp Ala Ala Ser Ala Thr
Leu Asn Glu Lys Ala Val -35 -30 -25 aaa gaa ttg aaa aaa gac ccg agc
gtc gct tac gtt gaa gaa gat cac 401 Lys Glu Leu Lys Lys Asp Pro Ser
Val Ala Tyr Val Glu Glu Asp His -20 -15 -10 gta gca cat gcg tac gcg
cag tcc gtg cct tac ggc gta tca caa att 449 Val Ala His Ala Tyr Ala
Gln Ser Val Pro Tyr Gly Val Ser Gln Ile -5 -1 1 5 10 aaa gcc cct
gct ctg cac tct caa ggc tac act gga tca aat gtt aaa 497 Lys Ala Pro
Ala Leu His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys 15 20 25 gta
gcg gtt atc gac agc ggt atc gat tct tct cat cct gat tta aag 545 Val
Ala Val Ile Asp Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys 30 35
40 gta gca ggc gga gcc agc atg gtt cct tct gaa aca nnn nnn ttc caa
593 Val Ala Gly Gly Ala Ser Met Val Pro Ser Glu Thr Xaa Xaa Phe Gln
45 50 55 gac nnn aac tct cac gga act cac gtt gcc ggc aca gtt gcg
gct ctt 641 Asp Xaa Asn Ser His Gly Thr His Val Ala Gly Thr Val Ala
Ala Leu 60 65 70 75 aat aac tca atc ggt gta tta ggc gtt gcg cca agc
nnn nnn ctt tac 689 Asn Asn Ser Ile Gly Val Leu Gly Val Ala Pro Ser
Xaa Xaa Leu Tyr 80 85 90 gct gta aaa gtt ctc ggt nnn nnn ggt tcc
ggc caa tac agc tgg atc 737 Ala Val Lys Val Leu Gly Xaa Xaa Gly Ser
Gly Gln Tyr Ser Trp Ile 95 100 105 att aac gga atc gag tgg gcg atc
gca aac aat atg gac gtt att aac 785 Ile Asn Gly Ile Glu Trp Ala Ile
Ala Asn Asn Met Asp Val Ile Asn 110 115 120 atg agc ctc ggc gga cct
tct ggt tct gct gct tta aaa gcg gca gtt 833 Met Ser Leu Gly Gly Pro
Ser Gly Ser Ala Ala Leu Lys Ala Ala Val 125 130 135 gat aaa gcc gtt
gca tcc ggc gtc gta gtc gtt gcg gca gcc ggt aac 881 Asp Lys Ala Val
Ala Ser Gly Val Val Val Val Ala Ala Ala Gly Asn 140 145 150 155 gaa
ggc nnn nnn ggc agc tca agc aca gtg ggc tac cct ggt aaa tac 929 Glu
Gly Xaa Xaa Gly Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr 160 165
170 cct tct gtc att gca gta ggc gct gtt gac agc agc aac caa aga gca
977 Pro Ser Val Ile Ala Val Gly Ala Val Asp Ser Ser Asn Gln Arg Ala
175 180 185 tct ttc tca agc gta gga cct gag ctt gat gtc atg gca cct
ggc gta 1025 Ser Phe Ser Ser Val Gly Pro Glu Leu Asp Val Met Ala
Pro Gly Val 190 195 200 tct atc caa agc acg ctt cct gga aac aaa tac
ggg gcg tac aac ggt 1073 Ser Ile Gln Ser Thr Leu Pro Gly Asn Lys
Tyr Gly Ala Tyr Asn Gly 205 210 215 acg tca atg gca tct ccg cac gtt
gcc gga gcg gct gct ttg att ctt 1121 Thr Ser Met Ala Ser Pro His
Val Ala Gly Ala Ala Ala Leu Ile Leu 220 225 230 235 tct aag cac ccg
aac tgg aca aac act caa gtc cgc agc agt tta nnn 1169 Ser Lys His
Pro Asn Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Xaa 240 245 250 aac
acc act aca aaa ctt ggt gat tct ttc tac tat gga aaa ggg ctg 1217
Asn Thr Thr Thr Lys Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu 255
260 265 atc aac gta cag gcg gca gct cag taa aacataaaaa accggccttg
1264 Ile Asn Val Gln Ala Ala Ala Gln 270 275 gccccgccgg tttttttatt
tttcttcctc cgcatgttca atccgctcca taatcgacgg 1324 atggctccct
ctgaaaattt taacgagaaa cggcgggttg acccggctca gtcccgtaac 1384
ggccaagtcc tgaaacgtct caatcgccgc ttcccggttt ccggtcagct caatgccgta
1444 acggtcggcg gcgttttcct gataccggga gacggcattc gtaatcggat c 1495
2 382 PRT Bacillus amyloliquefaciens VARIANT (1)...(1) Xaa = Met or
Val 2 Xaa Arg Gly Lys Lys Val Trp Ile Ser Leu Leu Phe Ala Leu Ala
Leu 1 5 10 15 Ile Phe Thr Met Ala Phe Gly Ser Thr Ser Ser Ala Gln
Ala Ala Gly 20 25 30 Lys Ser Asn Gly Glu Lys Lys Tyr Ile Val Gly
Phe Lys Gln Thr Met 35 40 45 Ser Thr Met Ser Ala Ala Lys Lys Lys
Asp Val Ile Ser Glu Lys Gly 50 55 60 Gly Lys Val Gln Lys Gln Phe
Lys Tyr Val Asp Ala Ala Ser Ala Thr 65 70 75 80 Leu Asn Glu Lys Ala
Val Lys Glu Leu Lys Lys Asp Pro Ser Val Ala 85 90 95 Tyr Val Glu
Glu Asp His Val Ala His Ala Tyr Ala Gln Ser Val Pro 100 105 110 Tyr
Gly Val Ser Gln Ile Lys Ala Pro Ala Leu His Ser Gln Gly Tyr 115 120
125 Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp Ser Gly Ile Asp Ser
130 135 140 Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala Ser Met Val
Pro Ser 145 150 155 160 Glu Thr Xaa Xaa Phe Gln Asp Xaa Asn Ser His
Gly Thr His Val Ala 165 170 175 Gly Thr Val Ala Ala Leu Asn Asn Ser
Ile Gly Val Leu Gly Val Ala 180 185 190 Pro Ser Xaa Xaa Leu Tyr Ala
Val Lys Val Leu Gly Xaa Xaa Gly Ser 195 200 205 Gly Gln Tyr Ser Trp
Ile Ile Asn Gly Ile Glu Trp Ala Ile Ala Asn 210 215 220 Asn Met Asp
Val Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Ala 225 230 235 240
Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala Ser Gly Val Val Val 245
250 255 Val Ala Ala Ala Gly Asn Glu Gly Xaa Xaa Gly Ser Ser Ser Thr
Val 260 265 270 Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala Val Gly
Ala Val Asp 275 280 285 Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val
Gly Pro Glu Leu Asp 290 295 300 Val Met Ala Pro Gly Val Ser Ile Gln
Ser Thr Leu Pro Gly Asn Lys 305 310 315 320 Tyr Gly Ala Tyr Asn Gly
Thr Ser Met Ala Ser Pro His Val Ala Gly 325 330 335 Ala Ala Ala Leu
Ile Leu Ser Lys His Pro Asn Trp Thr Asn Thr Gln 340 345 350 Val Arg
Ser Ser Leu Xaa Asn Thr Thr Thr Lys Leu Gly Asp Ser Phe 355 360 365
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala Ala Ala Gln 370 375 380
3 275 PRT Bacillus amyloliquefaciens 3 Ala Gln Ser Val Pro Tyr Gly
Val Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly Tyr
Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile
Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala 35 40 45 Ser
Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His 50 55
60 Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly
65 70 75 80 Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys
Val Leu 85 90 95 Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile
Asn Gly Ile Glu 100 105 110 Trp Ala Ile Ala Asn Asn Met Asp Val Ile
Asn Met Ser Leu Gly Gly 115 120 125 Pro Ser Gly Ser Ala Ala Leu Lys
Ala Ala Val Asp Lys Ala Val Ala 130 135 140 Ser Gly Val Val Val Val
Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly 145 150 155 160 Ser Ser Ser
Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala 165 170 175 Val
Gly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val 180 185
190 Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr
195 200 205 Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met
Ala Ser 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser
Lys His Pro Asn 225 230 235 240 Trp Thr Asn Thr Gln Val Arg Ser Ser
Leu Glu Asn Thr Thr Thr Lys 245 250 255 Leu Gly Asp Ser Phe Tyr Tyr
Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 4
275 PRT Bacillus subtilis 4 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln
Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser
Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser
His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro
Ser Glu Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr
His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80
Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85
90 95 Asp Ser Thr Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile
Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile Asn Met Ser
Leu Gly Gly 115 120 125 Pro Thr Gly Ser Thr Ala Leu Lys Thr Val Val
Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala Ala
Gly Asn Glu Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly
Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val
Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser
Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205
Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr 210
215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro
Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser
Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly
Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 5 274 PRT
Bacillus licheniformis 5 Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu
Ile Lys Ala Asp Lys Val 1 5 10 15 Gln Ala Gln Gly Phe Lys Gly Ala
Asn Val Lys Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Gln Ala Ser
His Pro Asp Leu Asn Val Val Gly Gly Ala 35 40 45 Ser Phe Val Ala
Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly 50 55 60 Thr His
Val Ala Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val 65 70 75 80
Leu Gly Val Ala Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn 85
90 95 Ser Ser Gly Ser Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu
Trp 100 105 110 Ala Thr Thr Asn Gly Met Asp Val Ile Asn Met Ser Leu
Gly Gly Ala 115 120 125 Ser Gly Ser Thr Ala Met Lys Gln Ala Val Asp
Asn Ala Tyr Ala Arg 130 135 140 Gly Val Val Val Val Ala Ala Ala Gly
Asn Ser Gly Asn Ser Gly Ser 145 150 155 160 Thr Asn Thr Ile Gly Tyr
Pro Ala Lys Tyr Asp Ser Val Ile Ala Val 165 170 175 Gly Ala Val Asp
Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly 180 185 190 Ala Glu
Leu Glu Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr 195 200 205
Pro Thr Asn Thr Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser Pro 210
215 220 His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn
Leu 225 230 235 240 Ser Ala Ser Gln Val Arg Asn Arg Leu Ser Ser Thr
Ala Thr Tyr Leu 245 250 255 Gly Ser Ser Phe Tyr Tyr Gly Lys Gly Leu
Ile Asn Val Glu Ala Ala 260 265 270 Ala Gln 6 269 PRT Homo sapiens
6 Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 1
5 10 15 His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu
Asp 20 25 30 Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly
Gly Ala Ser 35 40 45 Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly
Asn Gly His Gly Thr 50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu
Asn Asn Ser Ile Gly Val Leu 65 70 75 80 Gly Val Ala Pro Ser Ala Glu
Leu Tyr Ala Val Lys Val Leu Gly Ala 85 90 95 Ser Gly Ser Gly Ser
Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 100 105 110 Gly Asn Asn
Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 115 120 125 Pro
Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 130 135
140 Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser
145 150 155 160 Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala
Thr Asp Gln 165 170 175 Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly
Ala Gly Leu Asp Ile 180 185 190 Val Ala Pro Gly Val Asn Val Gln Ser
Thr Tyr Pro Gly Ser Thr Tyr 195 200 205 Ala Ser Leu Asn Gly Thr Ser
Met Ala Thr Pro His Val Ala Gly Ala 210 215 220 Ala Ala Leu Val Lys
Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile 225 230 235 240 Arg Asn
His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 245 250 255
Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260 265 7 15
PRT Artificial Sequence Description of Artificial Sequence
Synthetic 7 Ile Lys Asp Phe His Val Tyr Phe Arg Glu Ser Arg Asp Ala
Gly 1 5 10 15 8 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 8 Leu Glu Gln Ala Val Asn Ser Ala Thr
Ser Arg Gly Val Leu Val 1 5 10 15 9 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 9 Ala Gln Ser Val Pro
Trp Gly Ile Ser Arg Val Gln Ala Pro Ala 1 5 10 15 10 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 10
Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala His Asn 1 5 10
15 11 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 11 Gly Ile Ser Arg Val Gln Ala Pro Ala Ala His Asn Arg
Gly Leu 1 5 10 15 12 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 12 Arg Val Gln Ala Pro Ala Ala His
Asn Arg Gly Leu Thr Gly Ser 1 5 10 15 13 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 13 Ala Pro Ala Ala His
Asn Arg Gly Leu Thr Gly Ser Gly Val Lys 1 5 10 15 14 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 14
Ala His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val 1
5 10 15 15 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 15 Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala
Val Leu Asp Thr 1 5 10 15 16 15 PRT Artificial Sequence Description
of Artificial Sequence Synthetic 16 Thr Gly Ser Gly Val Lys Val Ala
Val Leu Asp Thr Gly Ile Ser 1 5 10 15 17 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 17 Gly Val Lys Val Ala
Val Leu Asp Thr Gly Ile Ser Thr His Pro 1 5 10 15 18 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 18
Val Ala Val Leu Asp Thr Gly Ile Ser Thr His Pro Asp Leu Asn 1 5 10
15 19 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 19 Leu Asp Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile
Arg Gly 1 5 10 15 20 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 20 Gly Ile Ser Thr His Pro Asp Leu
Asn Ile Arg Gly Gly Ala Ser 1 5 10 15 21 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 21 Thr His Pro Asp Leu
Asn Ile Arg Gly Gly Ala Ser Phe Val Pro 1 5 10 15 22 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 22
Asp Leu Asn Ile Arg Gly Gly Ala Ser Phe Val Pro Gly Glu Pro 1 5 10
15 23 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 23 Ile Arg Gly Gly Ala Ser Phe Val Pro Gly Glu Pro Ser
Thr Gln 1 5 10 15 24 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 24 Gly Ala Ser Phe Val Pro Gly Glu
Pro Ser Thr Gln Asp Gly Asn 1 5 10 15 25 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 25 Phe Val Pro Gly Glu
Pro Ser Thr Gln Asp Gly Asn Gly His Gly 1 5 10 15 26 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 26
Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr His Val 1 5 10
15 27 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 27 Ser Thr Gln Asp Gly Asn Gly His Gly Thr His Val Ala
Gly Thr 1 5 10 15 28 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 28 Asp Gly Asn Gly His Gly Thr His
Val Ala Gly Thr Ile Ala Ala 1 5 10 15 29 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 29 Gly His Gly Thr His
Val Ala Gly Thr Ile Ala Ala Leu Asn Asn 1 5 10 15 30 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 30
Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 1 5 10
15 31 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 31 Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val
Leu Gly 1 5 10 15 32 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 32 Ile Ala Ala Leu Asn Asn Ser Ile
Gly Val Leu Gly Val Ala Pro 1 5 10 15 33 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 33 Leu Asn Asn Ser Ile
Gly Val Leu Gly Val Ala Pro Ser Ala Glu 1 5 10 15 34 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 34
Ser Ile Gly Val Leu Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala 1 5 10
15 35 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 35 Val Leu Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val
Lys Val 1 5 10 15 36 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 36 Val Ala Pro Ser Ala Glu Leu Tyr
Ala Val Lys Val Leu Gly Ala 1 5 10 15 37 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 37 Ser Ala Glu Leu Tyr
Ala Val Lys Val Leu Gly Ala Ser Gly Ser 1 5 10 15 38 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 38
Leu Tyr Ala Val Lys Val Leu Gly Ala Ser Gly Ser Gly Ser Val 1 5 10
15 39 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 39 Val Lys Val Leu Gly Ala Ser Gly Ser Gly Ser Val Ser
Ser Ile 1 5 10 15 40 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 40 Leu Gly Ala Ser Gly Ser Gly Ser
Val Ser Ser Ile Ala Gln Gly 1 5 10 15 41 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 41 Ser Gly Ser Gly Ser
Val Ser Ser Ile Ala Gln Gly Leu Glu Trp 1 5 10 15 42 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 42
Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala Gly Asn 1 5 10
15 43 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 43 Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala Gly Asn Asn
Gly Met 1 5 10 15 44 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 44 Ala Gln Gly Leu Glu Trp Ala Gly
Asn Asn Gly Met His Val Ala 1 5 10 15 45 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 45 Leu Glu Trp Ala Gly
Asn Asn Gly Met His Val Ala Asn Leu Ser 1 5 10 15 46 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 46
Ala Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser 1 5 10
15 47 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 47 Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro
Ser Pro 1 5 10 15 48 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 48 His Val Ala Asn Leu Ser Leu Gly
Ser Pro Ser Pro Ser Ala Thr 1 5 10 15 49 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 49 Asn Leu Ser Leu Gly
Ser Pro Ser Pro Ser Ala Thr Leu Glu Gln 1 5 10 15 50 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 50
Leu Gly Ser Pro Ser Pro Ser Ala Thr Leu Glu Gln Ala Val Asn 1 5 10
15 51 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 51 Pro Ser Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser
Ala Thr 1 5 10 15 52 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 52 Ser Ala Thr Leu Glu Gln Ala Val
Asn Ser Ala Thr Ser Arg Gly 1 5 10 15 53 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 53 Leu Glu Gln Ala Val
Asn Ser Ala Thr Ser Arg Gly Val Leu Val 1 5 10 15 54 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 54
Ala Val Asn Ser Ala Thr Ser Arg Gly Val Leu Val Val Ala Ala 1 5 10
15 55 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 55 Ser Ala Thr Ser Arg Gly Val Leu Val Val Ala Ala Ser
Gly Asn 1 5 10 15 56 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 56 Ser Arg Gly Val Leu Val Val Ala
Ala Ser Gly Asn Ser Gly Ala 1 5 10 15 57 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 57 Val Leu Val Val Ala
Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile 1 5 10 15 58 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 58
Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser Tyr Pro 1 5 10
15 59 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 59 Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser Tyr Pro Ala
Arg Tyr 1 5 10 15 60 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 60 Ser Gly Ala Gly Ser Ile Ser Tyr
Pro Ala Arg Tyr Ala Asn Ala 1 5 10 15 61 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 61 Gly Ser Ile Ser Tyr
Pro Ala Arg Tyr Ala Asn Ala Met Ala Val 1 5 10 15 62 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 62
Ser Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr 1 5 10
15 63 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 63 Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp
Gln Asn 1 5 10 15 64 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 64 Ala Asn Ala Met Ala Val Gly Ala
Thr Asp Gln Asn Asn Asn Arg 1 5 10 15 65 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 65 Met Ala Val Gly Ala
Thr Asp Gln Asn Asn Asn Arg Ala Ser Phe 1 5 10 15 66 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 66
Gly Ala Thr Asp Gln Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr 1 5 10
15 67 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 67 Asp Gln Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly
Ala Gly 1 5 10 15 68 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 68 Asn Asn Arg Ala Ser Phe Ser Gln
Tyr Gly Ala Gly Leu Asp Ile 1 5 10 15 69 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 69 Ala Ser Phe Ser Gln
Tyr Gly Ala Gly Leu Asp Ile Val Ala Pro 1 5 10 15 70 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 70
Ser Gln Tyr Gly Ala Gly Leu Asp Ile Val Ala Pro Gly Val Asn 1 5 10
15 71 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 71 Gly Ala Gly Leu Asp Ile Val Ala Pro Gly Val Asn Val
Gln Ser 1 5 10 15 72 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 72 Leu Asp Ile Val Ala Pro Gly Val
Asn Val Gln Ser Thr Tyr Pro 1 5 10 15 73 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 73 Val Ala Pro Gly Val
Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr 1 5 10 15 74 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 74
Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr Ala Ser 1 5 10
15 75 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 75 Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr Ala Ser Leu
Asn Gly 1 5 10 15 76 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 76 Thr Tyr Pro Gly Ser Thr Tyr Ala
Ser Leu Asn Gly Thr Ser Met 1 5 10 15 77 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 77 Gly Ser Thr Tyr Ala
Ser Leu Asn Gly Thr Ser Met Ala Thr Pro 1 5 10 15 78 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 78
Tyr Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala 1 5 10
15 79 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 79 Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly
Ala Ala 1 5 10 15 80 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 80 Thr Ser Met Ala Thr Pro His Val
Ala Gly Ala Ala Ala Leu Val 1 5 10 15 81 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 81 Ala Thr Pro His Val
Ala Gly Ala Ala Ala Leu Val Lys Gln Lys 1 5 10 15 82 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 82
His Val Ala Gly Ala Ala Ala Leu Val Lys Gln Lys Asn Pro Ser 1 5 10
15 83 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 83 Gly Ala Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp
Ser Asn 1 5 10 15 84 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 84 Ala Leu Val Lys Gln Lys Asn Pro
Ser Trp Ser Asn Val Gln Ile 1 5 10 15 85 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 85 Lys Gln Lys Asn Pro
Ser Trp Ser Val Asn Gln Ile Arg Asn His 1 5 10 15 86 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 86
Asn Pro Ser Trp Ser Asn Val Gln Ile Arg Asn His Leu Lys Asn 1 5 10
15 87 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 87 Trp Ser Asn Val Gln Ile Arg Asn His Leu Lys Asn Thr
Ala Thr 1 5 10 15 88 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 88 Val Gln Ile Arg Asn His Leu Lys
Asn Thr Ala Thr Ser Leu Gly 1 5 10 15 89 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 89 Arg Asn His Leu Lys
Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn 1 5 10 15 90 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 90
Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu Tyr Gly 1 5 10
15 91 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 91 Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu Tyr Gly Ser
Gly Leu 1 5 10 15 92 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 92 Ser Leu Gly Ser Thr Asn Leu Tyr
Gly Ser Gly Leu Val Asn Ala 1 5 10 15 93 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 93 Ser Thr Asn Leu Tyr
Gly Ser Gly Leu Val Asn Ala Glu Ala Ala 1 5 10 15 94 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 94
Asn Leu Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 1 5 10
15 95 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 95 Asp Ala Glu Leu His Ile Phe Arg Val Phe Thr Asn Asn
Gln Val 1 5 10 15 96 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 96 Pro Leu Arg Arg Ala Ser Leu Ser
Leu Gly Ser Gly Phe Trp His 1 5 10 15 97 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 97 Arg Ala Ser Leu Ser
Leu Gly Ser Gly Phe Trp His Ala Thr Gly 1 5 10 15 98 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic 98
Leu Ser Leu Gly Ser Gly Phe Trp His Ala Thr Gly Arg His Ser 1 5 10
15 99 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 99 Gly Ser Gly Phe Trp His Ala Thr Gly Arg His Ser Ser
Arg Arg 1 5 10 15 100 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 100 Phe Trp His Ala Thr Gly Arg His
Ser Ser Arg Arg Leu Leu Arg 1 5 10 15 101 15 PRT Artificial
Sequence Description of Artificial Sequence Synthetic 101 Ala Thr
Gly Arg His Ser Ser Arg Arg Leu Leu Arg Ala Ile Pro 1 5 10 15 102
15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 102 Arg His Ser Ser Arg Arg Leu Leu Arg Ala Ile Pro Arg
Gln Val 1 5 10 15 103 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 103 Ser Arg Arg Leu Leu Arg Ala Ile
Pro Arg Gln Val Ala Gln Thr 1 5 10 15 104 15 PRT Artificial
Sequence Description of Artificial Sequence Synthetic 104 Leu Leu
Arg Ala Ile Pro Arg Gln Val Ala Gln Thr Leu Gln Ala 1 5 10 15 105
15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 105 Ala Ile Pro Arg Gln Val Ala Gln Thr Leu Gln Ala Asp
Val Leu 1 5
10 15 106 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 106 Arg Gln Val Ala Gln Thr Leu Gln Ala Asp Val
Leu Trp Gln Met 1 5 10 15 107 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 107 Ala Gln Thr Leu
Gln Ala Asp Val Leu Trp Gln Met Gly Tyr Thr 1 5 10 15 108 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
108 Leu Gln Ala Asp Val Leu Trp Gln Met Gly Tyr Thr Gly Ala Asn 1 5
10 15 109 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 109 Asp Val Leu Trp Gln Met Gly Tyr Thr Gly Ala
Asn Val Arg Val 1 5 10 15 110 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 110 Trp Gln Met Gly
Tyr Thr Gly Ala Asn Val Arg Val Ala Val Phe 1 5 10 15 111 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
111 Gly Tyr Thr Gly Ala Asn Val Arg Val Ala Val Phe Asp Thr Gly 1 5
10 15 112 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 112 Gly Ala Asn Val Arg Val Ala Val Phe Asp Thr
Gly Leu Ser Glu 1 5 10 15 113 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 113 Val Arg Val Ala
Val Phe Asp Thr Gly Leu Ser Glu Lys His Pro 1 5 10 15 114 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
114 Ala Val Phe Asp Thr Gly Leu Ser Glu Lys His Pro His Phe Lys 1 5
10 15 115 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 115 Asp Thr Gly Leu Ser Glu Lys His Pro His Phe
Lys Asn Val Lys 1 5 10 15 116 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 116 Leu Ser Glu Lys
His Pro His Phe Lys Asn Val Lys Glu Arg Thr 1 5 10 15 117 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
117 Lys His Pro His Phe Lys Asn Val Lys Glu Arg Thr Asn Trp Thr 1 5
10 15 118 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 118 His Phe Lys Asn Val Lys Glu Arg Thr Asn Trp
Thr Asn Glu Arg 1 5 10 15 119 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 119 Asn Val Lys Glu
Arg Thr Asn Trp Thr Asn Glu Arg Thr Leu Asp 1 5 10 15 120 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
120 Glu Arg Thr Asn Trp Thr Asn Glu Arg Thr Leu Asp Asp Gly Leu 1 5
10 15 121 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 121 Asn Trp Thr Asn Glu Arg Thr Leu Asp Asp Gly
Leu Gly His Gly 1 5 10 15 122 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 122 Asn Glu Arg Thr
Leu Asp Asp Gly Leu Gly His Gly Thr Phe Val 1 5 10 15 123 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
123 Thr Leu Asp Asp Gly Leu Gly His Gly Thr Phe Val Ala Gly Val 1 5
10 15 124 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 124 Asp Gly Leu Gly His Gly Thr Phe Val Ala Gly
Val Ile Ala Ser 1 5 10 15 125 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 125 Gly His Gly Thr
Phe Val Ala Gly Val Ile Ala Ser Met Arg Glu 1 5 10 15 126 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
126 Thr Phe Val Ala Gly Val Ile Ala Ser Met Arg Glu Cys Gln Gly 1 5
10 15 127 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 127 Ala Gly Val Ile Ala Ser Met Arg Glu Cys Gln
Gly Phe Ala Pro 1 5 10 15 128 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 128 Ile Ala Ser Met
Arg Glu Cys Gln Gly Phe Ala Pro Asp Ala Glu 1 5 10 15 129 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
129 Met Arg Glu Cys Gln Gly Phe Ala Pro Asp Ala Glu Leu His Ile 1 5
10 15 130 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 130 Cys Gln Gly Phe Ala Pro Asp Ala Glu Leu His
Ile Phe Arg Val 1 5 10 15 131 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 131 Phe Ala Pro Asp
Ala Glu Leu His Ile Phe Arg Val Phe Thr Asn 1 5 10 15 132 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
132 Asp Ala Glu Leu His Ile Phe Arg Val Phe Thr Asn Asn Gln Val 1 5
10 15 133 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 133 Leu His Ile Phe Arg Val Phe Thr Asn Asn Gln
Val Ser Tyr Thr 1 5 10 15 134 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 134 Phe Arg Val Phe
Thr Asn Asn Gln Val Ser Tyr Thr Ser Trp Phe 1 5 10 15 135 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
135 Phe Thr Asn Asn Gln Val Ser Tyr Thr Ser Trp Phe Leu Asp Ala 1 5
10 15 136 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 136 Asn Gln Val Ser Tyr Thr Ser Trp Phe Leu Asp
Ala Phe Asn Tyr 1 5 10 15 137 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 137 Ser Tyr Thr Ser
Trp Phe Leu Asp Ala Phe Asn Tyr Ala Ile Leu 1 5 10 15 138 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
138 Ser Trp Phe Leu Asp Ala Phe Asn Tyr Ala Ile Leu Lys Lys Ile 1 5
10 15 139 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 139 Leu Asp Ala Phe Asn Tyr Ala Ile Leu Lys Lys
Ile Asp Val Leu 1 5 10 15 140 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 140 Phe Asn Tyr Ala
Ile Leu Lys Lys Ile Asp Val Leu Asn Leu Ser 1 5 10 15 141 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
141 Ala Ile Leu Lys Lys Ile Asp Val Leu Asn Leu Ser Ile Gly Gly 1 5
10 15 142 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 142 Lys Lys Ile Asp Val Leu Asn Leu Ser Ile Gly
Gly Pro Asp Phe 1 5 10 15 143 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 143 Asp Val Leu Asn
Leu Ser Ile Gly Gly Pro Asp Phe Met Asp His 1 5 10 15 144 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
144 Asn Leu Ser Ile Gly Gly Pro Asp Phe Met Asp His Pro Phe Val 1 5
10 15 145 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 145 Ile Gly Gly Pro Asp Phe Met Asp His Pro Phe
Val Asp Lys Val 1 5 10 15 146 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 146 Pro Asp Phe Met
Asp His Pro Phe Val Asp Lys Val Trp Glu Leu 1 5 10 15 147 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
147 Met Asp His Pro Phe Val Asp Lys Val Trp Glu Leu Thr Ala Asn 1 5
10 15 148 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 148 Pro Phe Val Asp Lys Val Trp Glu Leu Thr Ala
Asn Asn Val Ile 1 5 10 15 149 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 149 Asp Lys Val Trp
Glu Leu Thr Ala Asn Asn Val Ile Met Val Ser 1 5 10 15 150 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
150 Trp Glu Leu Thr Ala Asn Asn Val Ile Met Val Ser Ala Ile Gly 1 5
10 15 151 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 151 Thr Ala Asn Asn Val Ile Met Val Ser Ala Ile
Gly Asn Asp Gly 1 5 10 15 152 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 152 Asn Val Ile Met
Val Ser Ala Ile Gly Asn Asp Gly Pro Leu Tyr 1 5 10 15 153 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
153 Met Val Ser Ala Ile Gly Asn Asp Gly Pro Leu Tyr Gly Thr Ile 1 5
10 15 154 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 154 Ala Ile Gly Asn Asp Gly Pro Leu Tyr Gly Thr
Leu Asn Asn Pro 1 5 10 15 155 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 155 Asn Asp Gly Pro
Leu Tyr Gly Thr Leu Asn Asn Pro Ala Asp Gln 1 5 10 15 156 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
156 Pro Leu Tyr Gly Thr Leu Asn Asn Pro Ala Asp Gln Met Asp Val 1 5
10 15 157 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 157 Gly Thr Leu Asn Asn Pro Ala Asp Gln Met Asp
Val Ile Gly Val 1 5 10 15 158 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 158 Asn Asn Pro Ala
Asp Gln Met Asp Val Ile Gly Val Gly Gly Ile 1 5 10 15 159 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
159 Ala Asp Gln Met Asp Val Ile Gly Val Gly Gly Ile Asp Phe Glu 1 5
10 15 160 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 160 Met Asp Val Ile Gly Val Gly Gly Ile Asp Phe
Glu Asp Asn Ile 1 5 10 15 161 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 161 Ile Gly Val Gly
Gly Ile Asp Phe Glu Asp Asn Ile Ala Arg Phe 1 5 10 15 162 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
162 Gly Gly Ile Asp Phe Glu Asp Asn Ile Ala Arg Phe Ser Ser Arg 1 5
10 15 163 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 163 Asp Phe Glu Asp Asn Ile Ala Arg Phe Ser Ser
Arg Gly Met Thr 1 5 10 15 164 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 164 Asp Asn Ile Ala
Arg Phe Ser Ser Arg Gly Met Thr Thr Trp Glu 1 5 10 15 165 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
165 Ala Arg Phe Ser Ser Arg Gly Met Thr Thr Trp Glu Leu Pro Gly 1 5
10 15 166 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 166 Ser Ser Arg Gly Met Thr Thr Trp Glu Leu Pro
Gly Gly Tyr Gly 1 5 10 15 167 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 167 Gly Met Thr Thr
Trp Glu Leu Pro Gly Gly Tyr Gly Arg Met Lys 1 5 10 15 168 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
168 Thr Trp Glu Leu Pro Gly Gly Tyr Gly Arg Met Lys Pro Asp Ile 1 5
10 15 169 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 169 Leu Pro Gly Gly Tyr Gly Arg Met Lys Pro Asp
Ile Val Thr Tyr 1 5 10 15 170 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 170 Gly Tyr Gly Arg
Met Lys Pro Asp Ile Val Thr Tyr Gly Ala Gly 1 5 10 15 171 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
171 Arg Met Lys Pro Asp Ile Val Thr Tyr Gly Ala Gly Val Arg Gly 1 5
10 15 172 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 172 Pro Asp Ile Val Thr Tyr Gly Ala Gly Val Arg
Gly Ser Gly Val 1 5 10 15 173 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 173 Val Thr Tyr Gly
Ala Gly Val Arg Gly Ser Gly Val Lys Gly Gly 1 5 10 15 174 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
174 Gly Ala Gly Val Arg Gly Ser Gly Val Lys Gly Gly Cys Arg Ala 1 5
10 15 175 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 175 Val Arg Gly Ser Gly Val Lys Gly Gly Cys Arg
Ala Leu Ser Gly 1 5 10 15 176 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 176 Ser Gly Val Lys
Gly Gly Cys Arg Ala Leu Ser Gly Thr Ser Val 1 5 10 15 177 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
177 Lys Gly Gly Cys Arg Ala Leu Ser Gly Thr Ser Val Ala Ser Pro 1 5
10 15 178 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 178 Cys Arg Ala Leu Ser Gly Thr Ser Val Ala Ser
Pro Val Val Ala 1 5 10 15 179 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 179 Leu Ser Gly Thr
Ser Val Ala Ser Pro Val Val Ala Gly Ala Val 1 5 10 15 180 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
180 Thr Ser Val Ala Ser Pro Val Val Ala Gly Ala Val Thr Leu Leu 1 5
10 15 181 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 181 Ala Ser Pro Val Val Ala Gly Ala Val Thr Leu
Leu Val Ser Thr 1 5 10 15 182 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 182 Val Val Ala Gly
Ala Val Thr Leu Leu Val Ser Thr Val Gln Lys 1 5 10 15 183 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
183 Gly Ala Val Thr Leu Leu Val Ser Thr Val Gln Lys Arg Glu Leu 1 5
10 15 184 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 184 Thr Leu Leu Val Ser Thr Val Gln Lys Arg Glu
Leu Val Asn Pro 1 5 10 15 185 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 185 Val Ser Thr Val
Gln Lys Arg Glu Leu Val Asn Pro Ala Ser Met 1 5 10 15 186 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
186 Val Gln Lys Arg Glu Leu Val Asn Pro Ala Ser Met Lys Gln Ala 1 5
10 15 187 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 187 Arg Glu Leu Val Asn Pro Ala Ser Met Lys Gln
Ala Leu Ile Ala 1 5 10 15 188 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 188 Val Asn Pro Ala
Ser Met Lys Gln Ala Leu Ile Ala Ser Ala Arg 1 5 10 15 189 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
189 Ala Ser Met Lys Gln Ala Leu Ile Ala Ser Ala Arg Arg Leu Pro 1 5
10 15 190 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 190 Lys Gln Ala Leu Ile Ala Ser Ala Arg Arg Leu
Pro Gly Val Asn 1 5 10 15 191 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 191 Leu Ile Ala Ser
Ala Arg Arg Leu Pro Gly Val Asn Met Phe Glu 1 5 10 15 192 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
192 Ser Ala Arg Arg Leu Pro Gly Val Asn Met Phe Glu Gln Gly His 1 5
10 15 193 15 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 193 Arg Leu Pro Gly Val Asn Met Phe Glu Gln Gly
His Gly Lys Leu 1 5 10 15 194 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 194 Gly Val Asn
Met Phe Glu Gln Gly His Gly Lys Leu Asp Leu Leu 1 5 10 15 195 15
PRT Artificial Sequence Description of Artificial Sequence
Synthetic 195 Met Phe Glu Gln Gly His Gly Lys Leu Asp Leu Leu Arg
Ala Tyr 1 5 10 15 196 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 196 Gln Gly His Gly Lys Leu Asp Leu
Leu Arg Ala Tyr Gln Ile Leu 1 5 10 15 197 15 PRT Artificial
Sequence Description of Artificial Sequence Synthetic 197 Gly Lys
Leu Asp Leu Leu Arg Ala Tyr Gln Ile Leu Asn Ser Tyr 1 5 10 15 198
15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 198 Asp Leu Leu Arg Ala Tyr Gln Ile Leu Asn Ser Tyr Lys
Pro Gln 1 5 10 15 199 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 199 Arg Ala Tyr Gln Ile Leu Asn Ser
Tyr Lys Pro Gln Ala Ser Leu 1 5 10 15 200 15 PRT Artificial
Sequence Description of Artificial Sequence Synthetic 200 Gln Ile
Leu Asn Ser Tyr Lys Pro Gln Ala Ser Leu Ser Pro Ser 1 5 10 15 201
15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 201 Asn Ser Tyr Lys Pro Gln Ala Ser Leu Ser Pro Ser Tyr
Ile Asp 1 5 10 15 202 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 202 Lys Pro Gln Ala Ser Leu Ser Pro
Ser Tyr Ile Asp Leu Thr Glu 1 5 10 15 203 15 PRT Artificial
Sequence Description of Artificial Sequence Synthetic 203 Ala Ser
Leu Ser Pro Ser Tyr Ile Asp Leu Thr Glu Cys Pro Tyr 1 5 10 15 204
15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 204 Ser Pro Ser Tyr Ile Asp Leu Thr Glu Cys Pro Tyr Met
Trp Pro 1 5 10 15 205 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 205 Tyr Ile Asp Leu Thr Glu Cys Pro
Tyr Met Trp Pro Tyr Cys Ser 1 5 10 15 206 15 PRT Artificial
Sequence Description of Artificial Sequence Synthetic 206 Leu Thr
Glu Cys Pro Tyr Met Trp Pro Tyr Cys Ser Gln Pro Ile 1 5 10 15 207
15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 207 Cys Pro Tyr Met Trp Pro Tyr Cys Ser Gln Pro Ile Tyr
Tyr Gly 1 5 10 15 208 1052 PRT Homo sapiens 208 Met Lys Leu Val Asn
Ile Trp Leu Leu Leu Leu Val Val Leu Leu Cys 1 5 10 15 Gly Lys Lys
His Leu Gly Asp Arg Leu Glu Lys Lys Ser Phe Glu Lys 20 25 30 Ala
Pro Cys Pro Gly Cys Ser His Leu Thr Leu Lys Val Glu Phe Ser 35 40
45 Ser Thr Val Val Glu Tyr Glu Tyr Ile Val Ala Phe Asn Gly Tyr Phe
50 55 60 Thr Ala Lys Ala Arg Asn Ser Phe Ile Ser Ser Ala Leu Lys
Ser Ser 65 70 75 80 Glu Val Asp Asn Trp Arg Ile Ile Pro Arg Asn Asn
Pro Ser Ser Asp 85 90 95 Tyr Pro Ser Asp Phe Glu Val Ile Gln Ile
Lys Glu Lys Gln Lys Ala 100 105 110 Gly Leu Leu Thr Leu Glu Asp His
Pro Asn Ile Lys Arg Val Thr Pro 115 120 125 Gln Arg Lys Val Phe Arg
Ser Leu Lys Tyr Ala Glu Ser Asp Pro Thr 130 135 140 Val Pro Cys Asn
Glu Thr Arg Trp Ser Gln Lys Trp Gln Ser Ser Arg 145 150 155 160 Pro
Leu Arg Arg Ala Ser Leu Ser Leu Gly Ser Gly Phe Trp His Ala 165 170
175 Thr Gly Arg His Ser Ser Arg Arg Leu Leu Arg Ala Ile Pro Arg Gln
180 185 190 Val Ala Gln Thr Leu Gln Ala Asp Val Leu Trp Gln Met Gly
Tyr Thr 195 200 205 Gly Ala Asn Val Arg Val Ala Val Phe Asp Thr Gly
Leu Ser Glu Lys 210 215 220 His Pro His Phe Lys Asn Val Lys Glu Arg
Thr Asn Trp Thr Asn Glu 225 230 235 240 Arg Thr Leu Asp Asp Gly Leu
Gly His Gly Thr Phe Val Ala Gly Val 245 250 255 Ile Ala Ser Met Arg
Glu Cys Gln Gly Phe Ala Pro Asp Ala Glu Leu 260 265 270 His Ile Phe
Arg Val Phe Thr Asn Asn Gln Val Ser Tyr Thr Ser Trp 275 280 285 Phe
Leu Asp Ala Phe Asn Tyr Ala Ile Leu Lys Lys Ile Asp Val Leu 290 295
300 Asn Leu Ser Ile Gly Gly Pro Asp Phe Met Asp His Pro Phe Val Asp
305 310 315 320 Lys Val Trp Glu Leu Thr Ala Asn Asn Val Ile Met Val
Ser Ala Ile 325 330 335 Gly Asn Asp Gly Pro Leu Tyr Gly Thr Leu Asn
Asn Pro Ala Asp Gln 340 345 350 Met Asp Val Ile Gly Val Gly Gly Ile
Asp Phe Glu Asp Asn Ile Ala 355 360 365 Arg Phe Ser Ser Arg Gly Met
Thr Thr Trp Glu Leu Pro Gly Gly Tyr 370 375 380 Gly Arg Met Lys Pro
Asp Ile Val Thr Tyr Gly Ala Gly Val Arg Gly 385 390 395 400 Ser Gly
Val Lys Gly Gly Cys Arg Ala Leu Ser Gly Thr Ser Val Ala 405 410 415
Ser Pro Val Val Ala Gly Ala Val Thr Leu Leu Val Ser Thr Val Gln 420
425 430 Lys Arg Glu Leu Val Asn Pro Ala Ser Met Lys Gln Ala Leu Ile
Ala 435 440 445 Ser Ala Arg Arg Leu Pro Gly Val Asn Met Phe Glu Gln
Gly His Gly 450 455 460 Lys Leu Asp Leu Leu Arg Ala Tyr Gln Ile Leu
Asn Ser Tyr Lys Pro 465 470 475 480 Gln Ala Ser Leu Ser Pro Ser Tyr
Ile Asp Leu Thr Glu Cys Pro Tyr 485 490 495 Met Trp Pro Tyr Cys Ser
Gln Pro Ile Tyr Tyr Gly Gly Met Pro Thr 500 505 510 Val Val Asn Val
Thr Ile Leu Asn Gly Met Gly Val Thr Gly Arg Ile 515 520 525 Val Asp
Lys Pro Asp Trp Gln Pro Tyr Leu Pro Gln Asn Gly Asp Asn 530 535 540
Ile Glu Val Ala Phe Ser Tyr Ser Ser Val Leu Trp Pro Trp Ser Gly 545
550 555 560 Tyr Leu Ala Ile Ser Ile Ser Val Thr Lys Lys Ala Ala Ser
Trp Glu 565 570 575 Gly Ile Ala Gln Gly His Val Met Ile Thr Val Ala
Ser Pro Ala Glu 580 585 590 Thr Glu Ser Lys Asn Gly Ala Glu Gln Thr
Ser Thr Val Lys Leu Pro 595 600 605 Ile Lys Val Lys Ile Ile Pro Thr
Pro Pro Arg Ser Lys Arg Val Leu 610 615 620 Trp Asp Gln Tyr His Asn
Leu Arg Tyr Pro Pro Gly Tyr Phe Pro Arg 625 630 635 640 Asp Asn Leu
Arg Met Lys Asn Asp Pro Leu Asp Trp Asn Gly Asp His 645 650 655 Ile
His Thr Asn Phe Arg Asp Met Tyr Gln His Leu Arg Ser Met Gly 660 665
670 Tyr Phe Val Glu Val Leu Gly Ala Pro Phe Thr Cys Phe Asp Ala Ser
675 680 685 Gln Tyr Gly Thr Leu Leu Met Val Asp Ser Glu Glu Glu Tyr
Phe Pro 690 695 700 Glu Glu Ile Ala Lys Leu Arg Arg Asp Val Asp Asn
Gly Leu Ser Leu 705 710 715 720 Val Ile Phe Ser Asp Trp Tyr Asn Thr
Ser Val Met Arg Lys Val Lys 725 730 735 Phe Tyr Asp Glu Asn Thr Arg
Gln Trp Trp Met Pro Asp Thr Gly Gly 740 745 750 Ala Asn Ile Pro Ala
Leu Asn Glu Leu Leu Ser Val Trp Asn Met Gly 755 760 765 Phe Ser Asp
Gly Leu Tyr Glu Gly Glu Phe Thr Leu Ala Asn His Asp 770 775 780 Met
Tyr Tyr Ala Ser Gly Cys Ser Ile Ala Lys Phe Pro Glu Asp Gly 785 790
795 800 Val Val Ile Thr Gln Thr Phe Lys Asp Gln Gly Leu Glu Val Leu
Lys 805 810 815 Gln Glu Thr Ala Val Val Glu Asn Val Pro Ile Leu Gly
Leu Tyr Gln 820 825 830 Ile Pro Ala Glu Gly Gly Gly Arg Ile Val Leu
Tyr Gly Asp Ser Asn 835 840 845 Cys Leu Asp Asp Ser His Arg Gln Lys
Asp Cys Phe Trp Leu Leu Asp 850 855 860 Ala Leu Leu Gln Tyr Thr Ser
Tyr Gly Val Thr Pro Pro Ser Leu Ser 865 870 875 880 His Ser Gly Asn
Arg Gln Arg Pro Pro Ser Gly Ala Gly Ser Val Thr 885 890 895 Pro Glu
Arg Met Glu Gly Asn His Leu His Arg Tyr Ser Lys Val Leu 900 905 910
Glu Ala His Leu Gly Asp Pro Lys Pro Arg Pro Leu Pro Ala Cys Pro 915
920 925 Arg Leu Ser Trp Ala Lys Pro Gln Pro Leu Asn Glu Thr Ala Pro
Ser 930 935 940 Asn Leu Trp Lys His Gln Lys Leu Leu Ser Ile Asp Leu
Asp Lys Val 945 950 955 960 Val Leu Pro Asn Phe Arg Ser Asn Arg Pro
Gln Val Arg Pro Leu Ser 965 970 975 Pro Gly Glu Ser Gly Ala Trp Asp
Ile Pro Gly Gly Ile Met Pro Gly 980 985 990 Arg Tyr Asn Gln Glu Val
Gly Gln Thr Ile Pro Val Phe Ala Phe Leu 995 1000 1005 Gly Ala Met
Val Val Leu Ala Phe Phe Val Val Gln Ile Asn Lys Ala 1010 1015 1020
Lys Ser Arg Pro Lys Arg Arg Lys Pro Arg Val Lys Arg Pro Gln Leu
1025 1030 1035 1040 Met Gln Gln Val His Pro Pro Lys Thr Pro Ser Val
1045 1050 209 280 PRT Homo sapiens 209 Arg Ala Ile Pro Arg Gln Val
Ala Gln Thr Leu Gln Ala Asp Val Leu 1 5 10 15 Trp Gln Met Gly Tyr
Thr Gly Ala Asn Val Arg Val Ala Val Phe Asp 20 25 30 Thr Gly Leu
Ser Glu Lys His Pro His Phe Lys Asn Val Lys Glu Arg 35 40 45 Thr
Asn Trp Thr Asn Glu Arg Thr Leu Asp Asp Gly Leu Gly His Gly 50 55
60 Thr Phe Val Ala Gly Val Ile Ala Ser Met Arg Glu Cys Gln Gly Phe
65 70 75 80 Ala Pro Asp Ala Glu Leu His Ile Phe Arg Val Phe Thr Asn
Asn Gln 85 90 95 Val Ser Tyr Thr Ser Trp Phe Leu Asp Ala Phe Asn
Tyr Ala Ile Leu 100 105 110 Lys Lys Ile Asp Val Leu Asn Leu Ser Ile
Gly Gly Pro Asp Phe Met 115 120 125 Asp His Pro Phe Val Asp Lys Val
Trp Glu Leu Thr Ala Asn Asn Val 130 135 140 Ile Met Val Ser Ala Ile
Gly Asn Asp Gly Pro Leu Tyr Gly Thr Leu 145 150 155 160 Asn Asn Pro
Ala Asp Gln Met Asp Val Ile Gly Val Gly Gly Ile Asp 165 170 175 Phe
Glu Asp Asn Ile Ala Arg Phe Ser Ser Arg Gly Met Thr Thr Trp 180 185
190 Glu Leu Pro Gly Gly Tyr Gly Arg Met Lys Pro Asp Ile Val Thr Tyr
195 200 205 Gly Ala Gly Val Arg Gly Ser Gly Val Lys Gly Gly Cys Arg
Ala Leu 210 215 220 Ser Gly Thr Ser Val Ala Ser Pro Val Val Ala Gly
Ala Val Thr Leu 225 230 235 240 Leu Val Ser Thr Val Gln Lys Arg Glu
Leu Val Asn Pro Ala Ser Met 245 250 255 Lys Gln Ala Leu Ile Ala Ser
Ala Arg Arg Leu Pro Gly Val Asn Met 260 265 270 Phe Glu Gln Gly His
Gly Lys Leu 275 280 210 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 210 Ile Lys Asp Phe His Val Tyr Phe
Arg glu Ser Arg Asp Ala Gly 1 5 10 15 211 15 PRT Artificial
Sequence Description of Artificial Sequence Synthetic 211 Asp Ala
Glu Leu His Ile Phe Arg Val Phe Thr Asn Asn Gln Val 1 5 10 15
* * * * *