U.S. patent application number 11/020628 was filed with the patent office on 2005-07-28 for proteins producing an altered immunogenic response and methods of making and using the same.
Invention is credited to Estell, David A., Harding, Fiona A..
Application Number | 20050164257 11/020628 |
Document ID | / |
Family ID | 34595738 |
Filed Date | 2005-07-28 |
United States Patent
Application |
20050164257 |
Kind Code |
A1 |
Estell, David A. ; et
al. |
July 28, 2005 |
Proteins producing an altered immunogenic response and methods of
making and using the same
Abstract
The present invention relates to a novel methods and
compositions for producing hyper and hypo allergenic compositions.
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.
Alternatively, T-cell epitopes are mutated to produce increased
immunogenic reactions. Moreover, naturally occurring proteins are
provided.
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: |
34595738 |
Appl. No.: |
11/020628 |
Filed: |
December 22, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11020628 |
Dec 22, 2004 |
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09677822 |
Oct 2, 2000 |
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09677822 |
Oct 2, 2000 |
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09500135 |
Feb 8, 2000 |
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6838269 |
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09500135 |
Feb 8, 2000 |
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09060872 |
Apr 15, 1998 |
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6835550 |
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Current U.S.
Class: |
435/6.16 ;
435/320.1; 435/372; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
G01N 33/6854 20130101;
C12N 9/54 20130101; C12Y 302/01004 20130101; A61K 38/00 20130101;
G01N 33/505 20130101; C12N 9/6424 20130101; C12N 9/20 20130101;
C12N 9/2437 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/372; 530/350; 536/023.5 |
International
Class: |
C12Q 001/68; C07H
021/04; C07K 014/74; C12N 005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 14, 1999 |
WO |
PCT/US99/08253 |
Claims
1. A variant of a polypeptide of interest comprising a T-cell
epitope, wherein said variant differs from said polypeptide of
interest by having an altered T-cell epitope such that said variant
produces an immunogenic response in an individual which is greater
than the immunogenic response produced by said polypeptide of
interest, wherein said T-cell epitope of said polypeptide of
interest is altered to produce said variant, and wherein said
polypeptide of interest is an enzyme selected from the group
consisting of lipase, cellulase, endo-glucosidase H, protease,
carbohydrases, reductase, oxidase, isomerase, transferase, kinase
and phosphatase.
2. (canceled)
3. (canceled)
4. (canceled)
5. The variant of claim 1 wherein said polypeptide of interest is
not recognized by said individual as endogenous to said
individual.
6. (canceled)
7. The variant of claim 1 wherein said T-cell epitope is altered
with amino acid substitutions.
8-28. (canceled)
29. A variant of a polypeptide of interest comprising at least one
T-cell epitope, wherein said variant differs from said polypeptide
of interest by having at least one altered T-cell epitope, such
that said variant produces an immunogenic response in an individual
which is greater than the immunogenic response produced by said
polypeptide of interest, wherein said at least one T-cell epitope
of said polypeptide of interest is altered to produce said variant
, and wherein said polypeptide of interest is an enzyme selected
from the group consisting of lipase, cellulase, endo-glucosidase H,
protease, carbohydrases, reductase, oxidase, isomerase,
transferase, kinase and phosphatase.
30. The variant of claim 29, wherein said polypeptide of interest
is not recognized by said individual as endogenous to said
individual.
31. The variant of claim 29, wherein said T-cell epitope is altered
with amino acid substitutions.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation in part of USSN
09/500,135, filed Apr. 2, 2000 which is a continuation in part of
USSN 09/060,872, filed on Apr. 15, 1998, both of which are
incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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 immunize 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.
[0011] 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.
[0012] While some studies have 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 involve measurement of IgE and
IgG antibody in blood sera previously exposed to the antigen.
However, 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. There is also a need to
produce proteins which produce an enhanced immunogenic response,
and a need to identify naturally occurring proteins which produce a
low immunogenic response. This invention meets these and other
needs.
SUMMARY OF THE INVENTION
[0013] The present invention provides proteins which produce
immunogenic responses as desired, methods of identifying and making
such proteins, and methods of using such proteins. For example, as
will be become apparent from the detailed description below, the
methods and compositions provided herein are useful in forming
hyper-and hypo-allergenic compositions. As used herein, hyper and
hypo means the composition produces a greater or lesser immunogenic
response, respectively, than the same composition without the
proteins of the present invention. Such compositions may include
cleaning compositions, textile treatments, contact lens cleaning
solutions or products, peptide hydrolysis products, waste treatment
products, cosmetic formulations including for skin, hair and oral
care, pharmaceuticals such as blood clot removal products, research
products such as enzymes and therapeutics including vaccines.
[0014] In one aspect of the invention, a polypeptide of interest is
selected and provided herein. The polypeptide of interest is
preferably one having a T-cell epitope and is then varied as
described below. However, polypeptides of interest may also be
selected based on naturally occuring properties and not altered.
Moreover, polypeptides of interest may be selected which do not
have a T-cell epitope, and altered so as to have a T-cell
epitope.
[0015] In one aspect of the invention provided herein is a variant
of a polypeptide of interest comprising a T-cell epitope. The
variant differs from the polypeptide of interest by having an
altered T-cell epitope such that said variant and said polypeptide
produce different immunogenic responses in an individual. The
variant can be prepared and selected to produce either a greater or
lesser immunogenic response than said polypeptide of interest.
[0016] The polypeptide of interest can be any polypeptide of
interest. In one aspect, the polypeptide is selected from the group
consisting of enzymes, hormones, factors, vaccines and cytokines.
In one embodiment, the polypeptide of interest is not recognized by
said individual as endogenous to said individual, or not recognized
as "self". As indicated herein, the polypeptide of interest may be
an enzyme. In one embodiment, the enzyme is selected from the group
consisting of lipase, cellulase, endo-glucosidase H, protease,
carbohydrase, reductase, oxidase, isomerase, transferase, kinase
and phosphatase. In preferred embodiments, the polypeptide of
interest and the variant of said polypeptide of interest each
comprise at least some of the same activity. For example, if a
variant of a protease is provided, said variant will produce an
altered immunogenic response, but will retain detectable, and
preferably comparable, protease activity.
[0017] Wherein a variant of a polypeptide of interest is provided,
the T-cell epitope may be altered in a number of ways including by
amino acid substitutions, deletions, additions and combinations
thereof. Preferably, the T-cell epitope is altered by having amino
acid substitutions. In one embodiment herein, the amino acid
substitutions are made to corresponding amino acids of a homolog of
the polypeptide of interest, wherein the homolog does not comprise
the same T-cell epitope in the corresponding position as the
polypeptide of interest. In one aspect, the terminal portion of the
polypeptide of interest comprising at least one T-cell epitope is
replaced with a corresponding terminal portion of the homolog of
the polypeptide of interest, wherein the replacement produces said
different immunogenic response.
[0018] In another embodiment provided herein, the nucleic acids
encoding the polypeptides producing the desired immunogenic
response are provided herein. Moreover, the invention includes
expression vectors and host cells comprising the nucleic acids
provided herein. Moreover, once the polypeptides and variants
thereof of the present invention are identified, substantially
homologous sequences of or those sequences which hybridize to the
polypeptides and variants can be identified and are provided
herein. Homologous is further defined below, and can refer to
similarity or identity, with identity being preferred. Preferably,
the homologous sequences are amino acid sequences or nucleic acids
encoding peptides having the activity of the polypeptides and
variants provided herein.
[0019] In yet another aspect of the invention is a method for
determining the immunogenic response produced by a protein. In one
embodiment, the method comprises (a) obtaining from a single blood
source a solution of dendritic cells and a solution of nave 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 nave CD4+and/or CD8+ T-cells with said
protein; and (d) measuring the proliferation of T-cells in said
step (c).
[0020] The methods of determining immunogenic responses produced by
proteins can also be used to identify comparative immunogenic
responses of proteins. Therefore, in one aspect, the method of
determining immunogenic responses of proteins further comprises
comparing immunogenic responses of one or more proteins. The
proteins can be homologs of each other, variants of the same
protein, different types of the same protein, for example,
different proteases, or different peptides of the same protein.
[0021] The invention further provides a method of altering the
immunogenicity of a polypeptide of interest comprising determining
the immunogenicity of said polypeptide; identifying a T-cell
epitope in a said polypeptide; and altering said T-cell epitope so
as to alter the immunogencity of said polypeptide. As described
herein, said altering can be done by altering a single amino acid
or switching a portion of the polypeptide of interest with a
corresponding portion of a homolog, wherein the switch produces an
altered immunogenic response.
[0022] Other aspects of the invention will be understood by the
skilled artisan by the following description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIGS. 1 A, 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.
[0024] 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).
[0025] 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'.
[0026] FIG. 4 illustrates the additive T-cell response of 16
peripheral mononuclear blood samples to peptides corresponding to
the Bacillus lentus protease (GG36). Peptide E05 includes the
region comprising residues corresponding to 170-173 in protease
from Bacillus amyloliquefaciens.
[0027] 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.
[0028] 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.
[0029] FIG. 7 illustrates the amino acid sequence of human
subtilisin (SEQ ID:NO 6).
[0030] FIG. 8 illustrates an amino acid sequence alignment of BPN'
(Bacillus amyloliquefaciens) protease, SAVINASE (Bacillus lentus)
protease and human subtilisin (S2HSBT).
[0031] 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.
[0032] 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.
[0033] FIG. 11 illustrates the T-cell response to various alanine
substitutions in the E05 protease peptide (an embodiment of the
T-cell epitope designated unmodified sequence) set in a sample
taken from an individual known to be hypersensitive to the
protease; the sequences for each peptide are also shown.
[0034] FIG. 12 illustrates the percent responders to the human
subtilisin molecule.
[0035] FIG. 13A illustrates the T-cell response of peptides derived
from Humicola insolens endogluconase (Accession number A23635).
Peptides A02 and F06 represent the region corresponding to residues
70-84 and 37-51, respectively, embodiments of the T-cell epitope,
of Humicola insolens endogluconase, wherein the full length
sequence is shown in FIG. 13B and A02 and F06 are shown underlined
and in bold.
[0036] FIG. 14A illustrates the T-cell response to peptides derived
from Thermomyces lanuginosa lipase (Accession number AAC08588 and
PID number g2997733). Peptides B02 and C06 represent the regions
corresponding to residues 83-100 and 108-121, respectively,
embodiments of the T-cell epitope, of Thermomyces lanuginosa
lipase, wherein the full length sequence is shown in FIG. 14B and
B02 and C06 are shown underlined and in bold.
[0037] FIG. 15A illustrates the T-cell response to peptides derived
from Streptomyces plicatus endo-beta-N-acetylglucosaminidase.
(Accession number P04067). Peptide C06 represents the region
corresponding to residues 126-140, an embodiment of the T-cell
epitope, of Streptomyces plicatus
endo-beta-N-acetylglucosaminidase, wherein the full length sequence
is shown in FIG. 15B and C06 is shown underlined and in bold.
[0038] FIG. 16 illustrates the T-cell response to peptides derived
from BPN' compiled for 22 individuals, wherein the sequences of
preferred T-cell epitopes are indicated.
[0039] FIG. 17 illustrates the T-cell response to peptides derived
from GG36 compiled for 22 individuals, wherein the sequences of
embodiments of T-cell epitopes are indicated, GSISYPARYANAMAVGA and
GAGLDIVAPGVNVQS being preferred.
[0040] FIG. 18 is an embodiment of a hybrid protein provided
herein, where the N-terminus comprises N-terminal GG36 sequence and
the C-terminus comprises C-terminal BPN' sequence, and wherein a
comparison of the sequences with those shown in FIG. 8 indicates
that the hybrid formed omits preferred T-cell epitopes of each
protein.
[0041] FIG. 19 is a comparison of ELISA titers for B.
amyloliquefaciens subtilisin and the same subtilisin but engineered
to contain a T-cell epitope from B. lentis subtilisin. FIG. 19a
represents the titer at 4 weeks; FIG. 19b at 6 weeks, FIG. 19c at 8
weeks and FIG. 19d at 10 weeks.
[0042] FIG. 20 is a time course study of ELISA titers for B.
amyloliquefaciens subtilisin and the same subtilisin but engineered
to contain a T-cell epitope from B. lentis subtilisin. FIG. 20a
represents the titer for a 1 .mu.g dose of enzyme, FIG. 20b a 5
.mu.g dose and FIG. 20c a 20 .mu.g dose.
DETAILED DESCRIPTION OF THE INVENTION
[0043] According to the present invention, a method for identifying
T-cell epitopes is provided. Moreover, proteins including naturally
occurring proteins which have relatively impotent or potent T-cell
epitopes or no T-cell epitopes may be identified in accordance with
the methods of the present invention. Thus, the present invention
allows the identification and production of proteins which produce
immunogenic responses as desired, including naturally occurring
proteins as well as proteins which have been mutated to produce the
appropriate response. It is understood that the terms protein,
polypeptide and peptide are sometimes used herein interchangeably.
Wherein a peptide is a portion of protein, the skilled artisan can
understand this by the context in which the term is used.
[0044] In one embodiment, the present invention provides an assay
which identifies epitopes and non-epitopes as follows:
differentiated dendritic cells are combined with nave 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 nave
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 nave CD4+ and/or CD8+
T-cells with a peptide of interest; (d) measuring the proliferation
of T-cells in said step (c).
[0045] In one embodiment, the peptide of interest to be analyzed is
derived from a polypeptide of interest. 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 preferred embodiment of the
invention, a series of peptide oligomers which correspond to all or
part of the polypeptide of interest are prepared. For example, a
peptide library is produced covering the relevant portion or all of
the protein. In one embodiment, the 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 greater reaction of one specific peptide
than its 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 different T-cell
response from that of the original protein. Alternatively, the
epitope may be used in its original form to stimulate an immune
response against a target, e.g. infectious agent or tumor cell.
Moreover, proteins may be identified herein which have desired high
or low T-cell epitope potency which may be used in their naturally
occurring forms.
[0046] "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.
[0047] "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.
[0048] "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.
[0049] "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).
[0050] "Sample" as used herein comprises mononuclear cells which
are nave, i.e., not sensitized, to the antigen in question.
[0051] "Homolog" as used herein means a protein or enzyme which has
similar catalytic action, structure and/or use as the protein of
interest. For purposes of this invention, a homolog and a protein
of interest are not necessarily related evolutionarily, e.g., same
functional protein from different species. 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.
[0052] 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.
[0053] The epitopes determined according to the assay provided
herein are then modified to reduce or augment the immunologic
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.
[0054] 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
immunogenic, e.g., 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 immunogenic, e.g., allergenic,
response due to T-cell epitope recognition than that of the protein
of interest; or (d) with any sequence which produces lesser
immunogenic, e.g., allergenic, response due to T-cell epitope
recognition than that of the protein of interest.
[0055] However, one of skill will readily recognize that epitopes
can be modified in other ways depending on the desired outcome. For
example, if a T-cell vaccine is desired, it is contemplated the
amino acid sequence of an epitope will be substituted with amino
acids which increase the immulogic response to the peptide via
enhanced MHC binding and/or T-cell recognition. In another example,
if altering an autoimmune response against self-antigens is
desired, it is contemplated the amino acid sequence of an epitope
will be substituted with amino acids that decrease or cause a shift
in an inflammatory or other immune response.
[0056] The present invention extends to all proteins against which
it is desired to modulate the immunogenic response, for example,
peptides to be used as T-cell vaccines, or peptides or proteins to
be used as therapeutic agents against, e.g., cancer, infectious
diseases and autoimmune diseases. One of skill in the art will
readily recognize the proteins and peptides of this invention are
not necessarily native proteins and peptides. Indeed, in one
embodiment of this invention, the assay described herein is used to
determine the immunologic response of proteins from shuffled genes.
For descriptions of gene shuffling and expression of such genes
see, Stemmer, Proc. Nat'l Acad. Sci. USA 91:10747 (1994); Patten,
et al., Current Opinion in Biotechnol. 8:724 (1997); Kuchner &
Arnold, Trends Biotechnol. 15:523 (1997); Moore, et al., J. Mol,
Biol. 272:336 (1997); Zhao, et al., Nature Biotechnol. 16:258
(1998); Giver, et al., Proc. Nat'l Acad. Sci. USA 95:12809 (1998);
Harayama, Trends Biotechnol. 16:76 (1998); Lin, et al.,
Biotechnol., Prog. 15:467 (1999); and Sun, J. Comput. Biol. 6:77
(1999). The assay is used to predict the immunologic response of
proteins encoded by shuffled genes. Once determined, the protein
can be altered to modulate the immunolgic response to that
protein.
[0057] In addition to the above proteins and peptides, the present
invention can be used to reduce the allergenicity of proteins.
These proteins include, but are not limited to, glucanases,
lipases, cellulases, endo-glucosidase Hs (endo-H), proteases,
carbohydrases, reductases, oxidases, isomerases, transferases,
kinases, phosphatases, amylases, etc. In addition to reducing the
allergenicity to an animal, such as a human, of naturally occurring
amino acid sequences, this invention encompasses reducing the
allergenicity of a mutated human protein, e.g., a protein that has
been altered to change the functional activity of the protein. In
many instances, the mutation of human proteins to e.g., increase
activity, results in the incorporation of new T-cell epitope in the
mutated protein. The assay of this invention can be used to
determine the presence of the new T-cell epitope and determine
substitute amino acids that will reduce the allergenicity of the
mutated protein. Although this invention encompasses the above
proteins and many others, 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 a-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.
[0058] In one embodiment herein, hybrid polypeptides are provided.
"Hybrid polypeptides" are proteins engineered from at least two
different proteins, which are preferably homologs of one another.
For example, a preferred hybrid polypeptide might have the
N-terminus of a protein and the C-terminus of a homolog of the
protein. In a preferred embodiment, the two terminal ends can be
combined to correspond to the full-length active protein. In a
preferred embodiment, the homologs share substantial similarity but
do not have identical T-cell epitopes. Therefore, in one
embodiment, for example, a polypeptide of interest having one or
more T-cell epitopes in the C-terminus may have the C-terminus
replaced with the C-terminus of a homolog having a less potent
T-cell epitope in the C-terminus, less T-cell epitopes, or no
T-cell epitope in the C-terminus. Thus, the skilled artisan
understands that by being able to identify T-cell epitopes among
homologs, a variety of variants producing different immunogenic
responses can be formed. Moreover, it is understood that internal
portions, and more than one homolog can be used to produce the
variants of the present invention.
[0059] More generally, the variants provided herein can be derived
from the precursor amino acid sequence by the substitution,
deletion, insertion, or combination thereof of one or more amino
acids of the precursor amino acid sequence. Such modification is
preferably of the "precursor DNA sequence" which encodes the amino
acid sequence of the precursor enzyme, but can be by the
manipulation of the precursor protein. 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 US patents
and applications already referenced herein).
[0060] 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.
[0061] "Recombinant", "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.
[0062] "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.
[0063] "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
proteins provided herein, including those from non-human sources
such as mouse or rabbit, which retain the essential activity of the
peptide, such as the ability to hydrolyze peptide bonds, etc., have
at least 50%, preferably at least 65% and most preferably at least
80%, more preferably at least 90%, and sometimes as much as 95 or
98% homology to the polypeptide of interest. In one embodiment, the
polypeptide of interest is shown in the Figures.
[0064] 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.
[0065] 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). "Corresponding" as used herein
generally refers to an analogous position along the peptide.
[0066] 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.
[0067] 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.
[0068] 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. W089/06279
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.
[0069] 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.
[0070] Homologous sequences can also be determined by using a
"sequence comparison algorithm." Optimal alignment of sequences for
comparison can be conducted, e.g., by the local homology algorithm
of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the
homology alignment algorithm of Needleman & Wunsch, J. Mol.
Biol. 48:443 (1970), by the search for similarity method of Pearson
& Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
visual inspection.
[0071] An example of an algorithm that is suitable for determining
sequence similarity is the BLAST algorithm, which is described in
Altschul, et al., J. Mol. Biol. 215:403-410 (1990). Software for
performing BLAST analyses is publicly available through the
National Center for Biotechnology Information
(http://www.ncbi.nim.nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence that either match or
satisfy some positive-valued threshold score T when aligned with a
word ofthe same length in a database sequence. These initial
neighborhood word hits act as starting points to find longer HSPs
containing them. The word hits are expanded in both directions
along each of the two sequences being compared for as far as the
cumulative alignment score can be increased. Extension of the word
hits is stopped when: the cumulative alignment score falls off by
the quantity X from a maximum achieved value; the cumulative score
goes to zero or below; or the end of either sequence is reached.
The BLAST algorithm parameters W, T, and X determine the
sensitivity and speed of the alignment. The BLAST program uses as
defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see
Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915
(1989)) alignments (B) of 50, expectation (E) of 10, M'5, N'-4, and
a comparison of both strands.
[0072] The BLAST algorithm then performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin &
Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, an amino acid
sequence is considered similar to a protein such as a protease if
the smallest sum probability in a comparison of the test amino acid
sequence to a protein such as a protease amino acid sequence is
less than about 0.1, more preferably less than about 0.01, and most
preferably less than about 0.001.
[0073] "Equivalent residues" may also be defined by determining
homology at the level of tertiary structure for a precursor protein
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 protein such as the
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 protein such as
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.
[0074] Equivalent residues which are functionally analogous to a
specific residue of Bacillus amyloliquefaciens subtilisin are
defined as those amino acids of the precursor protein such as a
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 protein, for example, 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 0.13 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.
[0075] 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 variants of the present invention include the mature forms of
protein variants, as well as the pro-and prepro-forms of such
protein variants. The prepro-forms are the preferred construction
since this facilitates the expression, secretion and maturation of
the protein variants.
[0076] "Prosequence" refers to a sequence of amino acids bound to
the N-terminal portion of the mature form of a protein which when
removed results in the appearance of the "mature" form of the
protein. 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 protein variants such as
protease variants is the putative prosequence of Bacillus
amyloliquefaciens subtilisin, although other prosequences may be
used.
[0077] A "signal sequence" or "presequence" refers to any sequence
of amino acids bound to the N-terminal portion of a protein or to
the N-terminal portion of a proprotein which may participate in the
secretion of the mature or pro forms of the protein. 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 protein gene which participate in the effectuation of the
secretion of protein under native conditions. The present invention
utilizes such sequences to effect the secretion of the protein
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).
[0078] A "prepro" form of a protein variant consists of the mature
form of the protein having a prosequence operably linked to the
amino terminus of the protein and a "pre" or "signal" sequence
operably linked to the amino terminus of the prosequence.
[0079] "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.
[0080] 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 protein is the
Bacillus strain BG2036 which is deficient in enzymatically active
neutral protein 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 protein include
Bacillus subtilis I 168 (also described in U.S. Pat. No. 4,760,025
(RE 34,606) and U.S. Pat. 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.
[0081] Host cells are transformed or transfected with vectors
constructed using recombinant DNA techniques. These techniques can
be found in any molecular biology practice guide, for example,
Sambrook et al. Molecular Cloning--A Laboratory Manual (2nd ed.)
Vol. 1-3, Cold Springs Harbor Publishing (1989) ("Sambrook"); and
Current Protocols in Molecular Biology, Ausubel et al.(eds.),
Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (1997 Supplement)
("Ausubel"). Such transformed host cells are capable of either
replicating vectors encoding the protein variants or expressing the
desired protein variant. In the case of vectors which encode the
pre-or prepro-form of the protein variant, such variants, when
expressed, are typically secreted from the host cell into the host
cell medium.
[0082] "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.
[0083] The genes encoding the naturally-occurring precursor protein
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
protein of interest, preparing genomic libraries from organisms
expressing the protein, and screening the libraries for the gene of
interest by hybridization to the probes. Positively hybridizing
clones are then mapped and sequenced.
[0084] "Hybridization" is used to analyze whether a given DNA
fragment or gene corresponds to a DNA sequence described herein and
thus falls within the scope of the present invention. Samples to be
hybridized are electrophoresed through an agarose gel (for example,
0.8% agarose) so that separation of DNA fragments can be visualized
by size. DNA fragments are typically visualized by ethidium bromide
staining. The gel may be briefly rinsed in distilled H2O and
subsequently depurinated in an appropriate solution (such as, for
example, 0.25M HCl) with gentle shaking followed by denaturation
for 30 minutes (in, for example, 0.4 M NaOH) with gentle shaking. A
renaturation step may be included, in which the gel is placed in
1.5 M NaCl, 1MTris, pH 7.0 with gentle shaking for 30 minutes.
[0085] The DNA should then be transferred onto an appropriate
positively charged membrane, for example, Maximum Strength Nytran
Plus membrane (Schleicher & Schuell, Keene, N.H.), using a
transfer solution (such as, for example, 6XSSC (900 mM NaCl, 90 mM
trisodium citrate). Once the transfer is complete, generally after
about 2 hours, the membrane is rinsed in e.g., 2X SSC (2X SSC =300
mM NaCl, 30 mM trisodium citrate) and air dried at room
temperature. The membrane should then be prehybridized (for
approximately 2 hours or more) in a suitable prehybridization
solution (such as, for example, an aqueous solution containing per
100 mL: 20-50 mL formamide, 25 mL of 20X SSPE (1X SSPE =0.18 M
NaCl, 1 mM EDTA, 10 mM NaH2PO4, pH 7.7), 2.5 mL of 20% SDS, and 1
mL of 10 mg/mL sheared herring or salmon sperm DNA). As would be
known to one of skill in the art, the amount of formamide in the
prehybridization solution may be varied depending on the nature of
the reaction obtained according to routine methods. Thus, a lower
amount of formamide may result in more complete hybridization in
terms of identifying hybridizing molecules than the same procedure
using a larger amount of formamide. On the other hand, a strong
hybridization band may be more easily visually identified by using
more formamide.
[0086] A DNA probe that is complementary or is nearly complementary
to the DNA sequence of interest and is generally between 100 and
1000 bases in length is labeled (using, for example, the Megaprime
labeling system according to the instructions of the manufacturer)
to incorporate 32P in the DNA. The labeled probe is denatured by
heating to 95.degree. C. for 5 minutes and immediately added to the
membrane and prehybridization solution. The hybridization reaction
should proceed for an appropriate time and under appropriate
conditions, for example, for 18 hours at 37.degree. C. with gentle
shaking or rotating. The membrane is rinsed (for example, in 2X
SSC/0.3% SDS) and then washed in an appropriate wash solution with
gentle agitation. The stringency desired will be a reflection of
the conditions under which the membrane (filter) is washed.
[0087] Specifically, the stringency of a given reaction (i.e., the
degree of homology necessary for successful hybridization) will
depend on the washing conditions to which the filter is subjected
after hybridization. "Low-stringency" conditions as defined herein
will comprise washing a filter with a solution of 0.2X SSC/0.1% SDS
at 20.degree. C. for 15 minutes. "High-stringency" conditions
comprise a further washing step comprising washing the filter a
second time with a solution of 0.2X SSC/0.1% SDS at 37.degree. C.
for 30 minutes.
[0088] After washing, the membrane is dried and the bound probe
detected. If 32P or another radioisotope is used as the labeling
agent, the bound probe can be detected by autoradiography. Other
techniques for the visualization of other probes are well-known to
those of skill. The detection of a bound probe indicates a nucleic
acid sequence has the desired homology and is encompassed within
this invention.
[0089] The cloned protein is then used to transform a host cell in
order to express the protein. The protein 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 protein gene
in certain eucaryotic host cells) which is exogenous or is supplied
by the endogenous terminator region of the protein 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 protein gene
into host genome. This is facilitated by procaryotic and eucaryotic
organisms which are particularly susceptible to homologous
recombination.
[0090] 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
protein may be produced. In such an approach, the DNA and/or amino
acid sequence of the precursor protein is determined. Multiple,
overlapping synthetic single-stranded DNA fragments are thereafter
synthesized, which upon hybridization and ligation produce a
synthetic DNA encoding the precursor protein. 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.
[0091] Once the naturally-occurring or synthetic precursor protein
gene has been cloned, a number of modifications are undertaken to
enhance the use of the gene beyond synthesis of the
naturally-occurring precursor protein. Such modifications include
the production of recombinant proteins as disclosed in U.S. Pat.
No. 4,760,025 (RE 34,606) and EPO Publication No. 0 251 446 and the
production of protein variants described herein.
[0092] The following cassette mutagenesis method may be used to
facilitate the construction of the protein variants of the present
invention, although other methods may be used. First, the
naturally-occurring gene encoding the protein is obtained and
sequenced in whole or 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 protein gene so as to facilitate the
replacement of the gene segment. However, any convenient
restriction site which is not overly redundant in the protein 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.
[0093] 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.
[0094] In one aspect of the invention, the objective is to secure a
variant protein having altered allergenic potential as compared to
the precursor protein, 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.
[0095] 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 protein will result in a the neutralization of a key T-cell
epitope in that protein. 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 protein 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 S103A/V104I/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.
[0096] 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/S103A/V104I/G159D/K170D/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/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
protein.
[0097] Based on the screening results obtained with the variant
proteins, 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.
[0098] Many of the protein variants of the invention are useful in
formulating various detergent compositions. A number of known
compounds are suitable surfactants useful in compositions
comprising the protein 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 protein variants of
the present invention may be used for any purpose that native or
wild-type proteins 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.
[0099] Proteins, particularly 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.
[0100] The addition of proteins, particularly 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 protein's denaturing
temperature. In addition, proteins of the invention can be used in
a cleaning composition without detergents, again either alone or in
combination with builders and stabilizers.
[0101] The variant proteins 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.
[0102] One aspect of the invention is a composition for the
treatment of a textile that includes variant proteins 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.
[0103] 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/Y171Q/S173D;
V68A/N76D/S103A/V104I/G159D/K17- 0D/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/Q245R-
/T260A of Bacillus amyloliquefaciens subtilisin.
[0104] The proteins of this invention exhibit modified
immunogenicity when compared to their precursor proteins. In
preferred embodiments, the proteins exhibit reduced allergenicity.
In other embodiments, the proteins exhibit increased
immunogenicity. The increase in immunogenicity is manifested by an
increase in B-cell or humoral immunological response, by an
increase in T-cell or cellular immunological response, or by an
increase in both B and T cell immunological responses. One of skill
will readily recognize that the uses of the proteins of this
invention will be determined, in large part, on the immunological
properties of the proteins. For example, enzymes that exhibit
reduced allergenicity can be used in cleaning compositions.
"Cleaning compositions" are compositions that can be used to remove
undesired compounds from substrates, such as fabric, dishes,
contact lenses, other solid substrates, hair (shampoos), skin
(soaps and creams), etc. Proteins, in particular, cellulases,
proteases, and amylases, with reduced allergenicity can also be
used in the treatment of textiles. "Textile treatment" comprises a
process wherein textiles, individual yarns or fibers that can be
woven, felted or knitted into textiles or garments are treated to
effect a desired characteristic. Examples of such desired
characteristics are "stone-washing" depilling, dehairing, desizing,
softening, and other textile treatments well known to those of
skill in the art.
[0105] Therapeutic proteins against which individuals mount an
immune response are also included in the invention. In particular,
individuals who lack endogenous production of the protein are
susceptible to forming neutralizing antibodies and become
refractile to treatment. Likewise, modifications of a protein may
introduce new epitopes that are potentially immunogeneic. Methods
of the invention can be used to identify and modify epitopes in,
e.g., human Factor VIII, to prevent neutralizing responses.
[0106] The pharmaceutical compositions can be prepared in various
forms, such as granules, tablets, pills, suppositories, capsules,
suspensions, salves, lotions and the like. Pharmaceutical grade
organic or inorganic carriers and/or diluents suitable for oral and
topical use can be used to make up compositions containing the
therapeutically_active compounds. Diluents known to the art include
aqueous media, vegetable and animal oils and fats. Stabilizing
agents, wetting and emulsifying agents, salts for varying the
osmotic pressure or buffers for securing an adequate pH value, and
skin penetration enhancers can be used as auxiliary agents. The
pharmaceutical compositions may also include one or more of the
following: carrier proteins such as serum albumin; buffers; fillers
such as microcrystalline cellulose, lactose, corn and other
starches; binding agents; sweeteners and other flavoring agents;
coloring agents; and polyethylene glycol. Additives are well known
in the art, and are used in a variety of formulations.
[0107] All publications and patents referenced herein are hereby
incorporated by reference in their entirety. The following is
presented by way of example and is not to be construed as a
limitation to the scope of the claims.
EXAMPLES
Example 1
Assay for the Identification of Peptide T-Cell Epitopes Using Nave
Human T-Cells
[0108] Fresh human peripheral blood cells were collected from
"nave" 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. Nave 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 mis 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.
[0109] From the resulting solution, a differentiated dendritic cell
culture was prepared from the peripheral blood mononuclear cell
sample having a density of 108 cells per 75 ml culture flask in a
solution as follows:
[0110] (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% CO2 to allow
adherence of monocytes to the flask wall.
[0111] (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% CO2. After five days, the cytokine TNFa
(Endogen) was added to 0.2 units/ml, and the cytokine IL-1a
(Endogen) was added to a final concentration of 50 units/ml and the
mixture incubated at 37.degree. C. in 5% CO2 for two more days.
[0112] (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% CO2. 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.
[0113] (4) The prepared dendritic cells were placed into a 96 well
round bottom array at 2.times.104/well in 100 microliter total
volume of AIM V media.
[0114] 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 108 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 600 G 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.
[0115] The CD4+ T-cell suspension was resuspended to a count of
2.times.106/ml in AIM V media to facilitate efficient manipulation
of the 96 well plate.
[0116] 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.
[0117] The final concentrations in each well, at 210 microliter
total volume are as follows:
[0118] 2.times.104 CD4+
[0119] 2.times.105 dendtritic cells (R:S of 10:1)
[0120] 5 mM peptide
Example 2
Identification of T-Cell Epitopes in Protease from Bacillus lentus
and Human subtilisin
[0121] 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, 15mers were
synthetically prepared, each 15mer overlapping with the previous
and the subsequent 15mer except for three residues.
[0122] 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.
[0123] 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. The greater the response, the more potent the T-cell
epitope is considered.
[0124] As indicated in FIGS. 4 and 5, the immunogenic response of
the nave 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.
[0125] 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-cells of a hypersensitive
individual.
[0126] 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, I 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.
[0127] From these results, it is apparent that the residues 170,
171 and 173 are largely responsible for the initiation of allergic
reaction within the protease from Bacillus lentus.
Example 3
Identification of T-Cell Epitopes in Cellulase from Humicola
insolens (Carezvme.RTM.)
[0128] The procedure described above was performed on peptides
derived from a cellulase from Humicola insolens (Carezyme .RTM.
from Novo Nordisk). As can be seen from FIG. 13, 2 T-cell epitopes
were discovered, A01 and F06.
Example 4
Identification of T-Cell Epitopes in Lioase from Thermomyces
Lanuginosa (Lipolase.RTM.)
[0129] The procedure described in Example 2 was performed on
peptides derived from a lipase from Thermomyces lanuginosa
(Lipolase .RTM. from Novo Nordisk). As can be seen from FIG. 14,
two T-cell epitopes were discovered, A12 and C06. Peptide E03
effected slightly increased T-cell proliferation in the nave
donors, however, this peptide is consecutive to A12 and they
represent one epitope. In this regard, the skilled artisan
understands that the length of the epitopes can be varied, and the
precise potency of the epitope, naturally occuring or mutated can
be determined by the methods herein.
Example 5
Identification of T-Cell Epitopes in Endoglucanase H from
Streptomyces plicatus
[0130] The procedure described in Example 2 was performed on
peptides derived from endoglucanase H from Streptomyces plicatus.
As can be seen from FIG. 15, a single T-cell epitope was
discovered, C06.
Example 6
Identification of T-Cell Epitopes in a Protease Hybrid
(GG36-BPN')
[0131] After determining the location of a T-cell epitope, a
protease hybrid was constructed using established protein
engineering techniques. The hybrid was constructed so that a highly
allergenic amino acid sequence of the protein was replaced with a
corresponding sequence from a less allergenic homolog. In this
instance, the first 122 amino acids of the protease were derived
from GG36, and the remaining amino acid sequence was derived from
BPN'.
[0132] The hybrid was first tested from a 100 ppm sample in North
American condition in 24 well assay at 0.5 ppm, superfixed
swatches, liquid (Tide KT) at 0.5 in 24 well assay with 3K
swatches, and in the N'N'-dimethyl Casein Assay, 5 g/l DMC in NA
detergent, TNBS dectection method.
[0133] The results are shown in FIGS. 16, 17 and 18.
Example 7
Identification of a Naturally Occuring Low Immunogenic Protein
[0134] Using the methods herein, proteinase K was identified as
producing a lower immunogenic response than other commercially
available proteases. Proteinase K as identified herein is from
Tritirachium Album limber. For a general description of proteases
and methodologies, see, Mathew, C.G.P. Isolation of high molecular
weight eukaryotic DNA, in Methods in Molecular Biology, vol. 2:
Nucleic Acids (Walker, J. M.,ed.), Humana, Clifton, N.J., (1984)
pp. 31-34.
Example 8:
T-cell Epitope Introduced into a Non-allergenic Protein
[0135] It has been found that Bacillus amyloliquefaciens subtilisin
is comparatively non-immunogenic when tested in Hartley strain
guinea pigs. A related protein from Bacillus lentis is highly
immunogenic. We had previously defined functional T cell epitopes
in the B. lentis molecule which were not found in the B.
amyloliquefaciens molecule, even though the sequences of interest
were highly homologous. In order to test the principle that the
presence of a functional T cell epitope can control the relative
levels of antibody production, we created a B. lentis-like T cell
epitope in the B. amyloliquefaciens molecule. This change was
accomplished by the substitution of a single amino acid in the B.
amyloliquefaciens sequence. B. amyloliquefaciens subtilisin and the
T cell epitope modified variant of B. amyloliquefaciens subtilisin
were tested in a guinea pig model of immungenicity.
[0136] B. lentis and B. amyloliquefaciens subtilisin T cell epitope
mapping: Guinea pigs were immunized with 20 .mu.g/immunization of
subtilisin from either B. lentis or B. amyloliquefaciens. Animals
were immunized subcutaneously in adjuvant every two weeks for 10 to
12 weeks. A single cell suspension of guinea pig splenocytes was
created from each animal's spleen. Cells were plated at
5.times.10.sup.5 splenocytes per well in round bottom 96 well
plates. 15-mer peptides off-set by 3 amino acids were synthesized
by Mimotopes. Peptides were resuspended to 1 mM in DMSO. Peptides
were added to the cells at a final concentration of 5 .mu.M.
Cultures were incubated for 5 days at 37 .degree., 5% CO.sub.2.
Wells were pulsed with 0.5 .mu.Ci tritiated thymidine, and allowed
to incubate for an additional 18 hours. Wells were harvested, and
thymidine incorporation assessed.
[0137] Two T cell epitopes were found in B. lentis subtilisin, and
none were found in B. amyloliquefaciens subtilisin (>10 animals
tested for these epitopes). The B. lentis T cell epitopes were
found to comprise the following sequences: IAALNNSIGVLGVAP (SEQ ID
NO:237) and LEWAGNNGMHVANLSLGS (SEQ ID NO:238)
[0138] For SEQ ID NO:237, the similar sequence in B.
amyloliquefaciens subtilisin is VAALNNSIGVLGVAP (SEQ ID NO:239).
The similar region in B. amyloliquefaciens subtilisin for SEQ ID
NO:238 was the much less homologous: IEWAIANNMDVINMSLG (SEQ ID
NO:240).
[0139] SEQ ID NO:237 and the homologous region in the B.
amyloliquefaciens subtilisin molecule (SEQ ID NO: 239) differ by
one amino acid: In B. lentis subtilisin the first amino acid is an
I, while it is a V in B. amyloliquefaciens. Therefore, we reasoned
that if we changed the V in the B. amyloliquefaciens sequence to an
I, we would create the B. lentis T cell epitope in the B.
amyloliquefaciens backbone.
[0140] This molecule was created by standard molecular biological
techniques, and was called B. amyloliquefaciens V72I. It was also
known as GP002.
[0141] Guinea Pig immunizations: Adult female Hartley guinea pigs
were immunized with various doses of B. amyloliquefaciens
subtilisin and GP002. The doses were 1, 5, 10, and 20 .mu.g/dose.
There were four animals for each dose. Animals were immunized
subcutaneously with enzyme in Complete Freund's Adjuvant for the
first immunization. All subsequent immunizations were performed in
Incomplete Freund's adjuvant. Animals were immunized, and a serum
sample taken, every two weeks.
[0142] ELISA: A direct ELISA was performed. Costart EIA plates were
coated with 10 .mu.g/ml of the immunizing enzyme in PBS overnight
at 4.degree. C. Plates were washed and blocked with 1% BSA in PBS.
Serum samples were diluted in 1% BSA/PBS, and incubated on the
enzymes coated plates for 1 hour. Serum samples were washed out,
and biotinylated anti-guinea pig IgG was added at a 1:10,000
dilution in 1% BSA/PBS. The secondary reagent was incubated for 1
hour. The wells were washed, and avidin conjugated horse radish
peroxidase was added to the wells at a 1:1000 dilution in 1%
BSA/PBS. After 30 minutes, the substrate (ABTS) was added and the
OD.sub.405 was read after 30 minutes.
[0143] Calculation of titers: Background was subtracted from the OD
readings, and the results plotted for each individual guinea pig. A
linear regression analysis was performed on the linear portion of
the curve. The titer value was calculated from the linear
regression equation for an OD =0.5. These individual titers were
then averaged.
[0144] Two guinea pigs in the 10 .mu.g dose of GP001 died at 2
weeks into the study. The data for the 10 .mu.g dose was therefore
thrown out.
[0145] Two results are immediately apparent: first, the GP002
variant increased the titers of antigen-specific antibody over the
entire time course for the lower doses of enzymes; and the GP002
variant increased titers of antigen-specific antibody for all doses
of enzymes in the earliest time points.
[0146] At the extended time points and for the higher doses, the
difference between B. amyloliquefaciens subtilisin and its variant
were no longer apparent. See FIGS. 19 and 20.
[0147] From the Figures it is apparent that a single change in the
amino acid sequence of B. amyloliquefaciens subtilisin
significantly altered its immunogenicity.
Sequence CWU 1
1
240 1 1495 DNA Bacillus amyloliquefaciens mat_peptide (417)..(1495)
CDS (96)..(1244) misc_feature (582)..(584) The nnn at positions 582
through 584 which in a preferred embodiment (aat) is to code for
asparagine, but which may also code for proline. 1 ggtctactaa
aatattattc catactatac aattaataca cagaataatc tgtctattgg 60
ttattctgca aatgaaaaaa aggagaggat aaaga atg aga ggc aaa aaa gta 113
Met 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 (163)...(163) Xaa = Asn or Pro 2
Met 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 Bacillus
lentus 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
Gly 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 Gly Ser Ile Ser Tyr Pro Ala Arg
Tyr Ala Asn Ala Met Ala Val 1 5 10 15 211 15 PRT Artificial
Sequence Description of Artificial Sequence Synthetic 211 Ala Ser
Ile Ser Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val 1 5 10 15 212
15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 212 Gly Ala Ile Ser Tyr Pro Ala Arg Tyr Ala Asn Ala Met
Ala Val 1 5 10 15 213 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 213 Gly Ser Ala Ser Tyr Pro Ala Arg
Tyr Ala Asn Ala Met Ala Val 1 5 10 15 214 15 PRT Artificial
Sequence Description of Artificial Sequence Synthetic 214 Gly Ser
Ile Ala Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val 1 5 10 15 215
15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 215 Gly Ser Ile Ser Ala Pro Ala Arg Tyr Ala Asn Ala Met
Ala Val 1 5 10 15 216 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 216 Gly Ser Ile Ser Tyr Ala Ala Arg
Tyr Ala Asn Ala Met Ala Val 1 5 10 15 217 15 PRT Artificial
Sequence Description of Artificial Sequence Synthetic 217 Gly Ser
Ile Ser Tyr Pro Ala Ala Tyr Ala Asn Ala Met Ala Val 1 5 10 15 218
15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 218 Gly Ser Ile Ser Tyr Pro Ala Arg Ala Ala Asn Ala Met
Ala Val 1 5 10 15 219 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 219 Gly Ser Ile Ser Tyr Pro Ala Arg
Tyr Ala Ala Ala Met Ala Val 1 5 10 15 220 15 PRT Artificial
Sequence Description of Artificial Sequence Synthetic 220 Gly Ser
Ile Ser Tyr Pro Ala Arg Tyr Ala Asn Ala Ala Ala Val 1 5 10 15 221
15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 221 Gly Ser Ile Ser Tyr Pro Ala Arg Tyr Ala Asn Ala Met
Ala Ala 1 5 10 15 222 15 PRT Humicola insolens 222 Pro Gly Gly Val
Ala Tyr Ser Cys Ala Asp Gln Thr Pro Trp Ala 1 5 10 15 223 15 PRT
Humicola insolens 223 Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln
Pro Val Phe Ser 1 5 10 15 224 276 PRT Humicola insolens 224 Met Arg
Ser Ser Pro Leu Leu Pro Ser Ala Val Val Ala Ala Leu Pro 1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Arg Ser Thr Arg Tyr Trp Asp Cys 20
25 30 Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln
Pro 35 40 45 Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Ile Thr Asp
Phe Asp Ala 50 55 60 Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr
Ser Cys Ala Asp Gln 65 70 75 80 Thr Pro Trp Ala Val Asn Asp Asp Phe
Ala Leu Gly Phe Ala Ala Thr 85 90 95 Ser Ile Ala Gly Ser Asn Glu
Ala Gly Trp Cys Cys Ala Cys Tyr Glu 100 105 110 Leu Thr Phe Thr Ser
Gly Pro Val Ala Gly Lys Lys Met Val Val Gln 115 120 125 Ser Thr Ser
Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn 130 135 140 Ile
Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe 145 150
155 160 Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn
Glu 165 170 175 Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr
Trp Arg Phe 180 185 190 Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe
Ser Phe Arg Gln Val 195 200 205 Gln Cys Pro Ala Glu Leu Val Ala Arg
Thr Gly Cys Arg Arg Asn Asp 210 215 220 Asp Gly Asn Phe Pro Ala Val
Gln Ile Pro Ser Ser Ser Thr Ser Ser 225 230 235 240 Pro Val Asn Gln
Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr 245 250 255 Ser Ser
Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu 260 265 270
Arg Trp Ala Gln 275 225 18 PRT Thermomyces lanuginosus 225 Gly Asp
Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile 1 5 10 15
Val Leu 226 15 PRT Thermomyces lanuginosus 226 Ser Ile Glu Asn Trp
Ile Gly Asn Leu Asn Phe Asp Leu Lys Glu 1 5 10 15 227 291 PRT
Thermomyces lanuginosus 227 Met Arg Ser Ser Leu Val Leu Phe Phe Val
Ser Ala Trp Thr Ala Leu 1 5 10 15 Ala Ser Pro Ile Arg Arg Glu Val
Ser Gln Asp Leu Phe Asn Gln Phe 20 25 30 Asn Leu Phe Ala Gln Tyr
Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn 35 40 45
Asp Ala Pro Ala Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro 50
55 60 Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp
Ser 65 70 75 80 Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn
Thr Asn Lys 85 90 95 Leu Ile Val Leu Ser Phe Arg Gly Ser Arg Ser
Ile Glu Asn Trp Ile 100 105 110 Gly Asn Leu Asn Phe Asp Leu Lys Glu
Ile Asn Asp Ile Cys Ser Gly 115 120 125 Cys Arg Gly His Asp Gly Phe
Thr Ser Ser Trp Arg Ser Val Ala Asp 130 135 140 Thr Leu Arg Gln Lys
Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr 145 150 155 160 Arg Val
Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val 165 170 175
Ala Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser 180
185 190 Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu
Thr 195 200 205 Val Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr
Asn Asp Ile 210 215 220 Val Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr
Ser His Ser Ser Pro 225 230 235 240 Glu Tyr Trp Ile Lys Ser Gly Thr
Leu Val Pro Val Thr Arg Asn Asp 245 250 255 Ile Val Lys Ile Glu Gly
Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro 260 265 270 Asn Ile Pro Asp
Ile Pro Ala His Leu Trp Tyr Phe Gly Leu Ile Gly 275 280 285 Thr Cys
Leu 290 228 15 PRT Streptomyces plicatus 228 Ile Lys Val Leu Leu
Ser Val Leu Gly Asn His Gln Gly Ala Gly 1 5 10 15 229 313 PRT
Streptomyces plicatus 229 Met Phe Thr Pro Val Arg Arg Arg Val Arg
Thr Ala Ala Leu Ala Leu 1 5 10 15 Ser Ala Ala Ala Ala Leu Val Leu
Gly Ser Thr Ala Ala Ser Gly Ala 20 25 30 Ser Ala Thr Pro Ser Pro
Ala Pro Ala Pro Ala Pro Ala Pro Val Lys 35 40 45 Gln Gly Pro Thr
Ser Val Ala Tyr Val Glu Val Asn Asn Asn Ser Met 50 55 60 Leu Asn
Val Gly Lys Tyr Thr Leu Ala Asp Gly Gly Gly Asn Ala Phe 65 70 75 80
Asp Val Ala Val Ile Phe Ala Ala Asn Ile Asn Tyr Asp Thr Gly Thr 85
90 95 Lys Thr Ala Tyr Leu His Phe Asn Glu Asn Val Gln Arg Val Leu
Asp 100 105 110 Asn Ala Val Thr Gln Ile Arg Pro Leu Gln Gln Gln Gly
Ile Lys Val 115 120 125 Leu Leu Ser Val Leu Gly Asn His Gln Gly Ala
Gly Phe Ala Asn Phe 130 135 140 Pro Ser Gln Gln Ala Ala Ser Ala Phe
Ala Lys Gln Leu Ser Asp Ala 145 150 155 160 Val Ala Lys Tyr Gly Leu
Asp Gly Val Asp Phe Asp Asp Glu Tyr Ala 165 170 175 Glu Tyr Gly Asn
Asn Gly Thr Ala Gln Pro Asn Asp Ser Ser Phe Val 180 185 190 His Leu
Val Thr Ala Leu Arg Ala Asn Met Pro Asp Lys Ile Ile Ser 195 200 205
Leu Tyr Asn Ile Gly Pro Ala Ala Ser Arg Leu Ser Tyr Gly Gly Val 210
215 220 Asp Val Ser Asp Lys Phe Asp Tyr Ala Trp Asn Pro Tyr Tyr Gly
Thr 225 230 235 240 Trp Gln Val Pro Gly Ile Ala Leu Pro Lys Ala Gln
Leu Ser Pro Ala 245 250 255 Ala Val Glu Ile Gly Arg Thr Ser Arg Ser
Thr Val Ala Asp Leu Ala 260 265 270 Arg Arg Thr Val Asp Glu Gly Tyr
Gly Val Tyr Leu Thr Tyr Asn Leu 275 280 285 Asp Gly Gly Asp Arg Thr
Ala Asp Val Ser Ala Phe Thr Arg Glu Leu 290 295 300 Tyr Gly Ser Glu
Ala Val Arg Thr Pro 305 310 230 15 PRT Bacillus amyloliquefaciens
230 Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly Val Leu Gly Val 1 5
10 15 231 15 PRT Bacillus amyloliquefaciens 231 Asn Gly Ile Glu Trp
Ala Ile Ala Asn Asn Met Asp Val Ile Asn 1 5 10 15 232 15 PRT
Bacillus lentus 232 Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp Thr
Gly Ile Ser 1 5 10 15 233 15 PRT Bacillus lentus 233 Ser Ala Glu
Leu Tyr Ala Val Lys Val Leu Gly Ala Ser Gly Ser 1 5 10 15 234 17
PRT Bacillus lentus 234 Gly Ser Ile Ser Tyr Pro Ala Arg Tyr Ala Asn
Ala Met Ala Val Gly 1 5 10 15 Ala 235 15 PRT Bacillus lentus 235
Gly Ala Gly Leu Asp Ile Val Ala Pro Gly Val Asn Val Gln Ser 1 5 10
15 236 272 PRT Artificial Sequence Description of Artificial
Sequence Hybrid of Bacillus lentus and Bacillus amyloliquefaciens
236 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 Ile Asn Met Ser Leu Gly Gly Ser Gly 115 120 125
Ser Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala Ser Gly Val 130
135 140 Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly Ser Ser
Ser 145 150 155 160 Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile
Ala Val Gly Ala 165 170 175 Val Asp Ser Ser Asn Gln Arg Ala Ser Phe
Ser Ser Val Gly Pro Glu 180 185 190 Leu Asp Val Met Ala Pro Gly Val
Ser Ile Gln Ser Thr Leu Pro Gly 195 200 205 Asn Lys Tyr Gly Ala Tyr
Asn Gly Thr Ser Met Ala Ser Pro His Val 210 215 220 Ala Gly Ala Ala
Ala Leu Ile Leu Ser Lys His Pro Asn Trp Thr Asn 225 230 235 240 Thr
Gln Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys Leu Gly Asp 245 250
255 Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala Ala Ala Gln
260 265 270 237 15 PRT Bacillus lentis subtilisin 237 Ile Ala Ala
Leu Asn Asn Ser Ile Gly Val Leu Gly Val Ala Pro 1 5 10 15 238 18
PRT Bacillus lentis subtilisin 238 Leu Glu Trp Ala Gly Asn Asn Gly
Met His Val Ala Asn Leu Ser Leu 1 5 10 15 Gly Ser 239 15 PRT
Bacillus amyloliquefaciens subtilisin 239 Val Ala Ala Leu Asn Asn
Ser Ile Gly Val Leu Gly Val Ala Pro 1 5 10 15 240 17 PRT Bacillus
amyloliquefaciens subtilisin 240 Ile Glu Trp Ala Ile Ala Asn Asn
Met Asp Val Ile Asn Met Ser Leu 1 5 10 15 Gly
* * * * *
References