U.S. patent application number 11/932411 was filed with the patent office on 2009-01-22 for inhibitors of human plasmin derived from the kunitz domains.
This patent application is currently assigned to Dyax Copr., a Delaware corporation. Invention is credited to Robert C. Ladner, William Markland.
Application Number | 20090023651 11/932411 |
Document ID | / |
Family ID | 26875521 |
Filed Date | 2009-01-22 |
United States Patent
Application |
20090023651 |
Kind Code |
A1 |
Markland; William ; et
al. |
January 22, 2009 |
INHIBITORS OF HUMAN PLASMIN DERIVED FROM THE KUNITZ DOMAINS
Abstract
This invention provides: novel proteins, which are homologous to
the first Kunitz domain (K1) of lipoprotein-associated coagulation
inhibitor (LACI), and which are capable of inhibiting plasmin; uses
of such novel proteins in therapeutic, diagnostic, and clinical
methods; and polynucleotides that encode such novel proteins.
Inventors: |
Markland; William;
(Southborough, MA) ; Ladner; Robert C.;
(Ijamsville, MD) |
Correspondence
Address: |
LOWRIE, LANDO & ANASTASI, LLP
ONE MAIN STREET, SUITE 1100
CAMBRIDGE
MA
02142
US
|
Assignee: |
Dyax Copr., a Delaware
corporation
|
Family ID: |
26875521 |
Appl. No.: |
11/932411 |
Filed: |
October 31, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11083742 |
Mar 18, 2005 |
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11932411 |
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10167351 |
Jun 11, 2002 |
6953674 |
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11083742 |
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09638770 |
Aug 15, 2000 |
6423498 |
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10167351 |
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09240136 |
Jan 29, 1999 |
6103499 |
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09638770 |
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08676124 |
Jan 7, 1997 |
6010880 |
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PCT/US95/00298 |
Jan 11, 1995 |
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09240136 |
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08208265 |
Mar 10, 1994 |
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08676124 |
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08179685 |
Jan 11, 1994 |
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08208265 |
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Current U.S.
Class: |
514/20.1 |
Current CPC
Class: |
C04B 35/5935 20130101;
A61P 7/04 20180101; C07K 14/8114 20130101; A61P 43/00 20180101;
A61P 7/00 20180101; A61P 35/04 20180101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 38/00 20060101
A61K038/00 |
Claims
1.-11. (canceled)
12. A method of treating liver cirrhosis or amyloidosis,
comprising: administering to a subject an effective amount of a
polypeptide comprising a non-naturally occurring Kunitz domain that
has the formula: TABLE-US-00021
Met-His-Ser-Phe-Cys-Ala-Phe-Lys-Ala-Xaa10-Xaa11-Gly-Xaa13-Cys-Xaa15--
Xaa16-
Xaa17-Xaa18-Xaa19-Arg-Trp-Xaa22-Xaa23-Asn-Ile-Phe-Thr-Arg-Gln-Cys-Xaa31-Xa-
a32-Phe-
Xaa34-Xaa35-Gly-Gly-Cys-Xaa39-Xaa40-Asn-Gln-Xaa43-Arg-Xaa45-Glu-Ser-Leu-Gl-
u-Glu- Cys-LysLys Met-Cys-Thr-Arg-Asp,
wherein Xaa10 is selected from the group consisting of Asp, Glu,
and Tyr; Xaa11 is selected from the group consisting of Thr, Ala,
Ser, Val, and Asp; Xaa13 is selected from the group consisting of
Pro, Leu, and Ala; Xaa15 is selected from the group consisting of
Arg and Lys; Xaa16 is selected from the group consisting of Ala and
Gly; Xaa17 is selected from the group consisting of Arg, Lys, and
Ser; Xaa18 is selected from the group consisting of Phe and Ile;
Xaa19 is selected from the group consisting of Glu, Asp, Pro, Gly,
Ser, and Ile; Xaa22 is selected from the group consisting of Tyr
and Phe; Xaa23 is selected from the group consisting of Tyr and
Phe; Xaa31 is selected from the group consisting of Asp, Glu, Thr,
Val, Gln, and Ala; Xaa32 is selected from the group consisting of
Thr, Ala, Glu, Pro, and Gln; Xaa34 is selected from the group
consisting of Val, Ile, Thr, Leu, Phe, Tyr, His, Asp, Ala, and Ser;
Xaa35 is selected from the group consisting of Tyr and Trp; Xaa39
is selected from the group consisting of Glu, Gly, Asp, Arg, Ala,
Gln, Leu, Lys, and Met; Xaa40 is selected from the group consisting
of Gly and Ala; Xaa43 is selected from the group consisting of Asn
and Gly; and Xaa45 is selected from the group consisting of Phe and
Tyr.
Description
[0001] The present application is a continuation-in-part of
application Ser. No. 08/208,265 (now pending) which in turn is a
continuation-in-part of Ser. No. 08/179,658, filed Jan. 11, 1994.
The entirety of each of these applications is hereby incorporated
by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to novel mutants of the first Kunitz
domain (K.sub.1) of the human lipoprotein-associated coagulation
inhibitor LACI, which inhibit plasmin. The invention also relates
to other modified Kunitz domains that inhibit plasmin and to other
plasmin inhibitors.
[0004] 2. Description of the Background Art
[0005] The agent mainly responsible for fibrinolysis is plasmin,
the activated form of plasminogen. Many substances can activate
plasminogen, including activated Hageman factor, streptokinase,
urokinase (uPA), tissue-type plasminogen activator (tPA), and
plasma kallikrein (pKA). pKA is both an activator of the zymogen
form of urokinase and a direct plasminogen activator.
[0006] Plasmin is undetectable in normal circulating blood, but
plasminogen, the zymogen, is present at about 3 .mu.M. An
additional, unmeasured amount of plasminogen is bound to fibrin and
other components of the extracellular matrix and cell surfaces.
Normal blood contains the physiological inhibitor of plasmin,
.alpha..sub.2-plasmin inhibitor (.alpha..sub.2-PI), at about 2
.mu.M. Plasmin and .alpha..sub.2-PI form a 1:1 complex. Matrix or
cell bound-plasmin is relatively inaccessible to inhibition by
.alpha..sub.2-PI. Thus, activation of plasmin can exceed the
neutralizing capacity of .alpha..sub.2-PI causing a profibrinolytic
state.
[0007] Plasmin, once formed: [0008] i. degrades fibrin clots,
sometimes prematurely; [0009] ii. digests fibrinogen (the building
material of clots) impairing hemostasis by causing formation of
friable, easily lysed clots from the degradation products, and
inhibition of platelet adhesion/aggregation by the fibrinogen
degradation products; [0010] iii. interacts directly with platelets
to cleave glycoproteins Ib and IIb/IIIa preventing adhesion to
injured endothelium in areas of high shear blood flow and impairing
the aggregation response needed for platelet plug formation
(ADEL86); [0011] iv. proteolytically inactivates enzymes in the
extrinsic coagulation pathway further promoting a prolytic
state.
[0012] Robbins (ROBB87) reviewed the plasminogen-plasmin system in
detail. ROBB87 and references cited therein are hereby incorporated
by reference.
Fibrinolysis and Fibrinogenolysis
[0013] Inappropriate fibrinolysis and fibrinogenolysis leading to
excessive bleeding is a frequent complication of surgical
procedures that require extracorporeal circulation, such as
cardiopulmonary bypass, and is also encountered in thrombolytic
therapy and organ transplantation, particularly liver. Other
clinical conditions characterized by high incidence of bleeding
diathesis include liver cirrhosis, amyloidosis, acute promyelocytic
leukemia, and solid tumors. Restoration of hemostasis requires
infusion of plasma and/or plasma products, which risks
immunological reaction and exposure to pathogens, e.g. hepatitis
virus and HIV.
[0014] Very high blood loss can resist resolution even with massive
infusion. When judged life-threatening, the hemorrhage is treated
with antifibrinolytics such as .epsilon.-amino caproic acid (See
HOOV93) (EACA), tranexamic acid, or aprotinin (NEUH89). Aprotinin
is also known as Trasylol.TM. and as Bovine Pancreatic Trypsin
Inhibitor (BPTI). Hereinafter, aprotinin will be referred to as
"BPTI". EACA and tranexamic acid only prevent plasmin from binding
fibrin by binding the kringles, thus leaving plasmin as a free
protease in plasma. BPTI is a direct inhibitor of plasmin and is
the most effective of these agents. Due to the potential for
thrombotic complications, renal toxicity and, in the case of BPTI,
immunogenicity, these agents are used with caution and usually
reserved as a "last resort" (PUTT89). All three of the
antifibrinolytic agents lack target specificity and affinity and
interact with tissues and organs through uncharacterized metabolic
pathways. The large doses required due to low affinity, side
effects due to lack of specificity and potential for immune
reaction and organ/tissue toxicity augment against use of these
antifibrinolytics prophylactically to prevent bleeding or as a
routine postoperative therapy to avoid or reduce transfusion
therapy. Thus, there is a need for a safe antifibrinolytic. The
essential attributes of such an agent are: [0015] i. Neutralization
of relevant target fibrinolytic enzyme(s); [0016] ii. High affinity
binding to target enzymes to minimize dose; [0017] iii. High
specificity for target, to reduce side effects; and [0018] iv. High
degree of similarity to human protein to minimize potential
immunogenicity and organ/tissue toxicity.
[0019] All of the fibrinolytic enzymes that are candidate targets
for inhibition by an efficacious antifibrinolytic are
chymotrypin-homologous serine proteases.
Excessive Bleeding
[0020] Excessive bleeding can result from deficient coagulation
activity, elevated fibrinolytic activity, or a combination of the
two conditions. In most bleeding diatheses one must control the
activity of plasmin. The clinically beneficial effect of BPTI in
reducing blood loss is thought to result from its inhibition of
plasmin (K.sub.D.about.0.3 nM) or of plasma kallikrein
(K.sub.D.about.100 nM) or both enzymes.
[0021] GARD93 reviews currently-used thrombolytics, saying that,
although thrombolytic agents (e.g. tPA) do open blood vessels,
excessive bleeding is a serious safety issue. Although tPA and
streptokinase have short plasma half lives, the plasmin they
activate remains in the system for a long time and, as stated, the
system is potentially deficient in plasmin inhibitors. Thus,
excessive activation of plasminogen can lead to a dangerous
inability to clot and injurious or fatal hemorrhage. A potent,
highly specific plasmin inhibitor would be useful in such
cases.
[0022] BPTI is a potent plasmin inhibitor, it has been found,
however, that it is sufficiently antigenic that second uses require
skin testing. Furthermore, the doses of BPTI required to control
bleeding are quite high and the mechanism of action is not clear.
Some say that BPTI acts on plasmin while others say that it acts by
inhibiting plasma kallikrein. FRAE89 reports that doses of about
840 mg of BPTI to 80 open-heart surgery patients reduced blood loss
by almost half and the mean amount transfused was decreased by 74%.
Miles Inc. has recently introduced Trasylol in USA for reduction of
bleeding in surgery (See Miles product brochure on Trasylol, which
is hereby incorporated by reference.) LOHM93 suggests that plasmin
inhibitors may be useful in controlling bleeding in surgery of the
eye. SHER89 reports that BPTI may be useful in limiting bleeding in
colonic surgery.
[0023] A plasmin inhibitor that is approximately as potent as BPTI
or more potent but that is almost identical to a human protein
domain offers similar therapeutic potential but poses less
potential for antigenicity.
Angiogenesis:
[0024] Plasmin is the key enzyme in angiogenesis. OREI94 reports
that a 38 kDa fragment of plasmin (lacking the catalytic domain) is
a potent inhibitor of metastasis, indicating that inhibition of
plasmin could be useful in blocking metastasis of tumors (FIDL94).
See also ELL192. ELL192, OREI94 and FIDL94 and the references cited
there are hereby incorporated by reference.
Plasmin
[0025] Plasmin is a serine protease derived from plasminogen. The
catalytic domain of plasmin (or "CatDom") cuts peptide bonds,
particularly after arginine residues and to a lesser extent after
lysines and is highly homologous to trypsin, chymotrypsin,
kallikrein, and many other serine proteases. Most of the
specificity of plasmin derives from the kringles' binding of fibrin
(LUCA83, VARA83, VARA84). On activation, the bond between
ARG.sub.561-Val.sub.562 is cut, allowing the newly free ammo
terminus to form a salt bridge. The kringles remain, nevertheless,
attached to the CatDom through two disulfides (COLM87, ROBB87).
[0026] BPTI has been reported to inhibit plasmin with K.sub.D of
about 300 pM (SCHN86). AUER88 reports that BPTI(R.sub.15) has
K.sub.i for plasmin of about 13 nM, suggesting that R.sub.15 is
substantially worse than K.sub.15 for plasmin binding. SCHN86
reports that BPTI in which the residues C.sub.14 and C.sub.3t have
been converted to Alanine has K.sub.i for plasmin of about 4.5 nM.
KIDO88 reports that APP-I has K.sub.i for plasmin of about 75 pM
(7.5.times.10.sup.-11 M), the most potent inhibitor of human
plasmin reported so far. DENN94a reports, however, that APP-I
inhibits plasmin with K.sub.i=225 nM (2.25.times.10.sup.-7 M). Our
second and third library were designed under the assumption that
APP-I is a potent plasmin binder. The selection process did not
select APP-I residues at most locations and the report of DENN94a
explains why this happened.
[0027] With recombinant DNA techniques, it is possible to obtain a
novel protein by expressing a mutated gene encoding a mutant of the
native protein gene. Several strategies for picking mutations are
known. In one strategy, some residues are kept constant, others are
randomly mutated, and still others are mutated in a predetermined
manner. This is called `variegation` and is defined in Ladner et
al. U.S. Pat. No. 5,223,409, which is incorporated by
reference.
[0028] DENN94a and DENN94b report selections of Kunitz domains
based on APP-I for binding to the complex of Tissue Factor with
Factor VII.sub.a. They did not use LACI-K1 as parental and did not
use plasmin as a target. The highest affinity binder they obtained
had K.sub.D for their target of about 2 nM. Our first-round
selectants have affinity in this range, but our second round
selectants are about 25-fold better than this.
[0029] Proteins taken from a particular species are assumed to be
less likely to cause an immune response when injected into
individuals of that species. Murine antibodies are highly antigenic
in humans. "Chimeric" antibodies having human constant domains and
murine variable domains are decidedly less antigenic. So called
"humanized" antibodies have human constant domains and variable
domains in which the CDRs are taken from murine antibodies while
the framework of the variable domains are of human origin.
"Humanized" antibodies are much less antigenic than are "chimeric"
antibodies. In a "humanized" antibody, fifty to sixty residues of
the protein are of non-human origin. The proteins of this invention
comprise, in most cases, only about sixty amino acids and usually
there are ten or fewer differences between the engineered protein
and the parental protein. Although humans do develop antibodies
even to human proteins, such as human insulins such antibodies tend
to bind weakly and the often do not prevent the injected protein
from displaying its intended biological function. Using a protein
from the species to be treated does not guarantee that there will
be no immune response. Nevertheless, picking a protein very close
in sequence to a human protein greatly reduces the risk of strong
immune response in humans.
[0030] Kunitz domains are highly stable and can be produced
efficiently in yeast or other host organisms. At least ten human
Kunitz domains have been reported. Although APP-1 was thought at
one time to be a potent plasmin inhibitor, there are, actually, no
human Kunitz domains that inhibit plasmin as well as does BPTI.
Thus, it is a goal of the present invention to provide sequences of
Kunitz domain that are both potent inhibitors of plasmin and close
in sequence to human Kunitz domains.
[0031] The use of site-specific mutagenesis, whether nonrandom or
random, to obtain mutant binding proteins of improved activity is
known in the art, but success is not assured.
SUMMARY OF THE INVENTION
[0032] This invention relates to mutants of BPTI-homologous Kunitz
domains that potently inhibit human plasmin. In particular, this
invention relates to mutants of one domain of human LACI which are
likely to be non-immunogenic to humans, and which inhibit plasmin
with K.sub.D, preferably, of about 5 nM or less, more preferably of
about 300 pM or less, and most preferably about 100 pM or less. The
invention also relates to the therapeutic and diagnostic use of
these novel proteins.
[0033] Plasmin-inhibiting proteins are useful for the prevention or
treatment of clinical conditions caused or exacerbated by plasmin,
including inappropriate fibrinolysis or fibrinogenolysis, excessive
bleeding associated with thrombolytics, post-operative bleeding,
and inappropriate androgenesis. Plasmin-binding mutants, whether or
not inhibitory, are useful for assaying plasmin in samples, in
vitro, for imaging areas of plasmin activity, in vivo, and for
purification of plasmin.
[0034] Preferred mutants QS4 and NS4 were selected from a library
that allowed about 50 million proteins having variability at
positions 13, 16, 17, 18, 19, 31, 32, 34, and 39. These proteins
have an amino-acid sequence nearly identical to a human protein but
inhibit plasmin with K.sub.i of about 2 nM (i.e. about 6-fold less
potent than BPTI, but 100-fold better than APP-I).
[0035] An especially preferred protein, SPI11, was selected from a
library allowing variability at positions 10, 11, 13, 15, 16, 17,
18, 19, and 21 and has an affinity for plasmin which is less than
100 .mu.M (i.e. about 3-fold superior to BPTI in binding), and yet
is much more similar in sequence to LACI, a human protein, than to
the BPTI, a bovine protein. Other LACI-K1 mutants selected from
this library and thought to have very high affinity for plasmin
include SPI15, SPI08, and SPI23. An additional library allowing
variation at positions 10, 11, 13, 15, 16, 17, 18, 19, 21, 31, 32,
34, 35, and 39 has been screened and a consensus sequence (SPIcon1)
found. Variants shown to be better than QS4, and thus more
preferred, include SPI51 and SPI47. Sequences that are likely to
have very high affinity for plasmin yet retain an essentially human
amino-acid sequence have been identified and include sequences
SPI60, SPI59, SPI42, SPI55, SPI56, SPI52, SPI46, SPI49, SPI53,
SPI41, and SPI57. The amino-acid sequence information that confers
high affinity for the active site of plasmin can be transferred to
other Kunitz domains, particularly to Kunitz domains of human
origin; designs of several such proteins are disclosed.
[0036] The preferred plasmin inhibitors of the present invention
fulfill one or more of the following desiderata: [0037] 1) the
K.sub.i for plasmin is at most 20 nM, preferably not more than
about 5 nM, more preferably not more than about 300 pM, and most
preferably, not more than about 100 pM, [0038] 2) the inhibitor
comprises a Kunitz domain meeting the requirements shown in Table
14 with residues number by reference to BPTI, [0039] 3) at the
Kunitz domain positions 12-21 and 32-39 one of the amino-acid types
listed for that position-in Table 15, and [0040] 4) the inhibitor
is more similar in amino-acid sequence to a reference sequence
selected from the group SPI11, SPI15, SPI08, SPI23, SPI51, SPI47,
QS4, NS4, Human LACI-K2, Human LACI-K3, Human collagen .alpha.3
KuDom, Human TFPI-2 DOMAIN 1, Human TFPI-2 DOMAIN 2, Human TFPI-2
DOMAIN 3, HUMAN ITI-K1, Human ITI-K2, HUMAN PROTEASE NEXIN-II,
Human APP-I, DPI-1.1.1, DPI-1.1.2, DPI-1.1.3, DPI-1.2.1, DPI-1.3.1,
DPI-2.1, DPI-3.1.1, DPI-3.2.1, DPI-3.3.1, DPI-4.1.1, DPI-4.2.1,
DPI-4.2.2, DPI-4.2.3, DPI-4.2.4, DPI-4.2.5, DPI-5.1, DPI-5.2,
DPI-6.1, DPI-6.2 than is the amino acid sequence of said Kunitz
domain to the sequence of BPTI.
Nomenclature
[0041] Herein, affinities are stated as K.sub.D
(K.sub.D(A,B)=[A][B]/[A-B]). A numerically smaller K.sub.D reflects
higher affinity. For the purposes of this invention, a "plasmin
inhibiting protein" is one that binds and inhibits plasmin with
K.sub.i of about 20 nM or less. "Inhibition" refers to blocking the
catalytic activity of plasmin and so is measurable in vitro in
assays using chromogenic or fluorogenic substrates or in assays
involving macromolecules.
[0042] Amino-acid residues are discussed in three ways: full name
of the amino acid, standard three-letter code, and standard
single-letter code. Table use only the one-letter code. The text
uses full names and three-letter code where clarity requires.
TABLE-US-00001 A = Ala C = Cys D = Asp E = Glu F = Phe G = Gly H =
His I = Ile K = Lys L = Leu M = Met N = Asn P = Pro Q = Gln R = Arg
S = Ser T = Thr V = Val W = Trp Y = Tyr
[0043] For the purposed of this invention, "substantially
homologous" sequences are at least 51%, more preferably at least
80%, identical, over any specified regions. Herein, sequences that
are identical are understood to be "substantially homologous".
Sequences would still be "substantially homologous" if within one
region of at least 20 amino acids they are sufficiently similar
(51% or more) but outside the region of comparison they differed
totally. An insertion of one amino acid in one sequence relative to
the other counts as one mismatch. Most preferably, no more than six
residues, other than at termini, are different. Preferably, the
divergence in sequence, particularly in the specified regions, is
in the form of "conservative modifications".
[0044] "Conservative modifications" are defined as [0045] (a)
conservative substitutions of amino acids as defined in Table 9;
and [0046] (b) single or multiple insertions or deletions of amino
acids at termini, at domain boundaries, in loops, or in other
segments of relatively high mobility.
[0047] Preferably, except at termini, no more than about six amino
acids are inserted or deleted at any locus, and the modifications
are outside regions known to contain important binding sites.
Kunitz Domains
[0048] Herein, "Kunitz domain" and "KuDom" are used interchangeably
to mean a homologue of BPTI (not of the Kunitz soya-bean trypsin
inhibitor). A KuDom is a domain of a protein having at least 51
amino acids (and up to about 61 amino acids) containing at least
two, and preferably three, disulfides. Herein, the residues of all
Kunitz domains are numbered by reference to BPTI (i.e. residues
1-58). Thus the first cysteine residue is residue 5 and the last
cysteine is 55. An amino-acid sequence shall, for the purposed of
this invention, be deemed a Kunitz domain if it can be aligned,
with three or fewer mismatches, to the sequence shown in Table 14.
An insertion or deletion of one residue shall count as one
mismatch. In Table 14, "x" matches any amino acid and "X" matches
the types listed for that position. Disulfides bonds link at least
two of: 5 to 55, 14 to 38, and 30 to 51. The number of disulfides
may be reduced by one, but none of the standard cysteines shall be
left unpaired. Thus, if one cysteine is changed, then a
compensating cysteine is added in a suitable location or the
matching cysteine is also replaced by a non-cysteine (the latter
being generally preferred). For example, Drosophila funebris male
accessory gland protease inhibitor has no cysteine at position 5,
but has a cysteine at position-1 (just before position 1);
presumably this forms a disulfide to CYS.sub.55. If Cys.sub.14 and
Cys.sub.38 are replaced, the requirement of Gly.sub.12, (Gly or
Ser).sub.37, and Gly.sub.36 are dropped. From zero to many
residues, including additional domains (including other KuDoms),
can be attached to either end of a Kunitz domain.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0049] Protease inhibitors, such as Kunitz domains, function by
binding into the active site of the protease so that a peptide bond
(the "scissile bond") is: 1) not cleaved, 2) cleaved very slowly,
or 3) cleaved to no effect because the structure of the inhibitor
prevents release or separation of the cleaved segments. In Kunitz
domains, disulfide bonds act to hold the protein together even if
exposed peptide bonds are cleaved. From the residue on the amino
side of the scissile bond, and moving away from the bond, residues
are conventionally called P1, P2, P3, etc. Residues that follow the
scissile bond are called P1', P2', P3', etc. (SCHE67, SCHE68). It
is generally accepted that each serine protease has sites
(comprising several residues) S1, S2, etc. that receive the side
groups and main-chain atoms of residues P1, P2, etc. of the
substrate or inhibitor and sites S1', S2', etc. that receive the
side groups and main-chain atoms of P1', P2', etc. of the substrate
or inhibitor. It is the interactions between the S sites and the P
side groups and main chain atoms that give the protease specificity
with respect to substrates and the inhibitors specificity with
respect to proteases. Because the fragment having the new amino
terminus leaves the protease first, many worker designing small
molecule protease inhibitors have concentrated on compounds that
bind sites S1, S2, S3, etc.
[0050] LASK80 reviews protein protease inhibitors. Some inhibitors
have several reactive sites on one polypeptide chain, and these
domains usually have different sequences, specificities, and even
topologies. It is known that substituting amino acids in the
P.sub.5 to P.sub.5' region influences the specificity of an
inhibitor. Previously, attention has been focused on the P1 residue
and those very close to it because these can change the specificity
from one enzyme class to another. LASK80 suggests that among
KuDoms, inhibitors with P1=Lys or Arg inhibit trypsin, those with
P1=Tyr, Phe, Trp, Leu and Met inhibit chymotrypsin, and those with
P1=Ala or Ser are likely to inhibit elastase. Among the Kazal
inhibitors, LASK80 continues, inhibitors with P1=Leu or Met are
strong inhibitors of elan, and in the Bowman-Kirk family elastase
is inhibited with P1=Ala, but not with P1=Leu. Such limited changes
do not provide inhibitors of truly high affinity (i.e. better than
1 to 10 nM).
[0051] Kunitz domains are defined above. The 3D structure (at high
resolution) of BPTI (the archetypal Kunitz domain) is known. One of
the X-ray structures is deposited in the Brookhaven Protein Data
Bank as "6PTI"]. The 3D structure of some BPTI homologues (EIGE90,
HYNE90) are known. At least seventy KuDom sequences are known.
Known human homologues include three KuDoms of LACI (WUNT88,
GIRA89, NOVO89), two KuDoms of inter-.alpha.-Trypsin Inhibitor,
APP-I (KIDO88), a KuDom from collagen, and three KuDoms of TFPI-2
(SPRE94).
LACI
[0052] Lipoprotein-associated coagulation inhibitor (LACI) is a
human serum phosphoglycoprotein with a molecular weight of 39 kDa
(amino-acid sequence in Table 1) containing three KuDoms. We refer
hereinafter to the protein as LACI and to the Kunitz domains
thereof as LACI-K1 (residues 50 to 107), LACI-K2 (residues 121 to
178), and LACI-K3 (213 to 270). The cDNA sequence of LACI is
reported in WUNT88. GIRA89 reports mutational studies in which the
P1 residues of each of the three KuDoms were altered. LACI-K1
inhibits Factor VIIa (F.VII.sub.a) when F.VII.sub.a is complexed to
tissue factor and LACI-K2 inhibits Factor X.sub.a. It is not known
whether LACI-K3 inhibits anything. Neither LACI nor any of the
KuDoms of LACI is a potent plasmin inhibitor.
[0053] KuDoms of this invention are substantially homologous with
LACI-K1, but differ in ways that confer strong plasmin inhibitory
activity discussed below. Other KuDoms of this invention are
homologous to other naturally-occurring KuDoms, particularly to
other human KuDoms. For use in humans, the proteins of this
invention are designed to be more similar in sequence to a human
KuDom than to BPTI, to reduce the risk of causing an immune
response.
First Library of LACI-K1 and Selectants for Binding to Plasmin
[0054] Applicants have screened a first library of LACI-K1 for
mutants having high affinity for human plasmin and obtained the
sequences shown in Table 2 and Table 3. These sequences may be
summarized as shown in Table 16, where "preferred residues" are
those appearing in at least one of the 32 variants identified as
binding plasmin. The preferences at residues 13, 16, 17, 18 and 19
are strong, as shown in Table 17. Although the range of types
allowed at 31 and 32 is limited, the selection indicates that an
acidic group at 31 and a neutral group at 32 is preferred. At
residue 17, Arg was preferred; Lys, another positively charged
amino acid, was not in the library, and may be a suitable
substitute for Arg. Many amino-acid types at positions 34 and 39
are consistent with high-affinity plasmin binding, but some types
may hinder binding.
[0055] It should be appreciated that Applicants have not sequenced
all the positive isolates of this or other libraries herein
disclosed, and that some of the possible proteins may not have been
present in detectable amounts.
[0056] Applicants have prepared one of the selected proteins, QS4,
shown in Table 2. QS4 inhibits plasmin with a K.sub.i of about 2
nM. Although this level of inhibition is less than that of BPT1,
QS4 is a preferred molecule for use in humans because it has less
potential for immunogenicity. Other proteins shown in Table 2 and
Table 3 are very likely to be potent inhibitors of plasmin and are
likely to pose little threat of antigenicity.
Second Library that Varies Residues 10-21
[0057] Applicants have prepared a second library of LACI-K1
derivatives shown in Table 5 and allowing variation at residues 10,
11, 13, 15, 16, 17, 18, 19, and 21. This was screened for binding
to plasmin and the proteins shown in Table 6 were obtained.
[0058] "Consensus" in Table 6 is E.sub.10TGPCRARFERW.sub.21, where
the seven underscored residues differ from LACI-K1. Only acidic
amino acids (Glu:17 or Asp:15) were seen at position 10; Lys and
Asn are not acceptable. As Glu and Asp appeared with almost equal
frequency, they probably to contribute equally to binding. Acidic
residues were not seen at position 11. Thr was most common (11/32)
with Ser appearing often (9/32); Gly appeared 8 times. At 13, Pro
was strongly preferred (24/32) with Ala second at 5/32. At 15, Arg
was strongly preferred (25/32), but a few (7/32) isolates have Lys.
Note that BPTI(R.sub.15) is a worse plasmin inhibitor than is BPTI.
At 16, Ala was preferred (22/32), but Gly did appeared fairly often
(10/32). At 17, Arg was most common (15/32), with Lys coming second
(9/32). At residues 17 and 18, APP-I has Met and Ile. At 18, we
allowed Ile or Phe. Only four isolates have Ile at 18 and none of
these have Met at 17. This was surprising in view of KIDO88, but
quite understandable in view of DENN94a. This collection of
isolates has a broad distribution at 19: (Glu:8, Pro:7, Asp:4,
Ala:3. His:3, Gly:2, Gln:2, Asn:1, Ser:1, and Arg:1), but acidic
side groups are strongly preferred over basic ones. At 21, the
distribution was (Trp:16, Phe:14, Leu:2, Cys:0); BPTI has Tyr at
21.
[0059] The binding of clonally pure phage that display one or
another of these proteins was compared to the binding of BPTI phage
(Table 6). Applicants have determined the K.sub.i of protein SPI11
and found it to be about 88 pM which is substantially superior to
BPTI.
Third Library that Varies 10-21 and 31-39
[0060] Applicants used a pool of phage of the second library
(varied at residues 10, 11, 13, 15, 16, 17, 18, 19, and 21) that
had been selected twice for plasmin binding as a source of DNA into
which variegation was introduced at residues 31, 32, 34, 35, and 39
as shown in Table 7.
[0061] This library was screened for three rounds for binding to
plasmin and the isolates shown in Table 8 were obtained. The
distribution of amino-acid types is shown in Table 18 where "x"
means the amino-acid type was not allowed and "*" indicates the
wild-type for LACI-K1.
[0062] These sequences gave a consensus in the 10-21 and 31-40
region of E.sub.10TGPCRAKFDRW.sub.21 . . . E.sub.31AFVYGGCGG.sub.40
(SPIcon1 in Table 4). The ten underscored amino acids differ from
LACI-K1. At eight varied positions, a second type was quite common:
Asp at 10, Ala at 11, Glu at 19, Phe at 21, Thr at 31; Pro or Ser
it 32, Leu or Ile at 34, and Glu at 39. At position 17, the highly
potent inhibitor SPI11 has R. Thus, the sequence
D.sub.10TGPCRARFDRF.sub.21 . . . E.sub.31AFIYGGCEG.sub.40
(DPI-1.1.1 in Table 4) differs from LACI-K1 by only six residues,
matches the selected sequences at the residues having strong
consensus, and has preferred substitutions at positions 10, 17, 21,
34, and 39. DPI-1.1.1 is expected to have a very high affinity for
plasmin and little potential for immunogenicity in humans.
[0063] Preliminary testing of proteins SPI11, BPTI, SPI23, SPI51,
SPI47, QS4, SPI22, SPI54, and SPI43 for plasmin inhibitory activity
placed them in the order given. SPI11 is significantly more potent
than BPTI with K.sub.i of about 88 pM. SPI23 and SPI51 are very
similar in activity and only slightly less potent than BPTI. SPI47
is less potent than SPI15 but better than QS4. SPI22 is weaker than
QS4. SPI54 and SPI43 are not so potent as QS4, K.sub.i probably
>4 nM.
[0064] A KuDom that is highly homologous at residues 5-55 to any
one of the sequences SPI11, SPI15, SPI08, SPI23, SPI51, SPI47, QS4,
and NS4, as shown in Table 4, is likely to be a potent inhibitor
(K.sub.D>5 nM) of plasmin and have a low potential for
antigenicity in humans. More preferably, to have high affinity for
plasmin, a KuDom would have a sequence that is identical at
residues 10-21 and 31-39 and has five or fewer differences at
residues 5-9, 22-30, and 40-55 as compared to any of the sequences
SPI11, SPI15, SPI08, SPI23, SPI51, SPI47, QS4, and NS4.
[0065] Using the selected sequences and the binding data of
selected and natural KuDoms, we can write a recipe for a
high-affinity plasmin-inhibiting KuDom that can be applied to other
human KuDom parentals. First, the KuDom must meet the requirements
in Table 14. The substitutions shown in Table 15 are likely to
confer high-affinity plasmin inhibitory activity on any KuDom. Thus
a protein that contains a sequence that is a KuDom, as shown in
Table 14, and that contains at each of the position 12-21 and 32-39
an amino-acid type shown in Table 15 for that position is likely to
be a potent inhibitor of human plasmin. More preferably, the
protein would have an amino-acid type shown in Table 15 for all of
the positions listed in Table 15. To reduce the potential for
immune response, one should use one or another human KuDom as
parental protein to give the sequence outside the binding
region.
[0066] It is likely that a protein that comprises an amino-acid
sequence that is substantially homologous to SPI11 from residue 5
through residue 55 (as shown in Table 4) and is identical to SPI11
at positions 13-19, 31, 32, 34, and 39 will inhibit human plasmin
with a K.sub.i of 5 nM or less. SPI11 differs from LACI-K1 at 7
positions. It is not clear that these substitutions are equally
important in fostering plasmin binding and inhibition. There are
seven molecules in which one of the substituted positions of SPI11
is changed to the residue found in LACI-K1 (i.e. "reverted"), 21 in
which two of the residues are reverted, 35 in which three residues
are reverted, 35 in which four are reverted, 21 in which five are
reverted, and seven in which six are reverted. It is expected that
those with more residues reverted will have less affinity for
plasmin but also less potential for immunogenicity. A person
skilled in the art can pick a protein of sufficient potency and low
immunogenicity from this collection of 126. It is also possible
that substitutions in SPI11 by amino acids that differ from LACI-K1
can reduce the immunogenicity without reducing the affinity for
plasmin to a degree that makes the protein unsuitable for use as a
drug.
Designed KuDom Plasmin Inhibitors
[0067] Hereinafter, "DPI" will mean a "Designed Plasmin Inhibitor"
that are KuDoms that incorporate amino-acid sequence information
from the SPI series of molecules, especially SPI11. Sequences of
several DPIs and their parental proteins are given in Table 4.
[0068] Sequences DPI-1.1.1, DPI-1.1.2, DPI-1.1.3, DPI-1.1.4,
DPI-1.1.5, and DPI-1.1.6 (in Table 4) differ from LACI-K1 by 6, 5,
5, 4, 3, and 2 amino acids respectively and represent a series in
which affinity for plasmin may decrease slowly while similarity to
a human sequence increases so as to reduce likelihood of
immunogenicity. The selections from each of the libraries show that
M18F is a key substitution and that either I17K or I17R is very
important. Selections from the second and third library indicate
that Arg is strongly preferred at 15, that an acid side group at 11
is disadvantageous to binding. The highly potent inhibitor SPI11
differs from the consensus by having R.sub.17, as does BPTI.
DPI-1.1.1 carries the mutations D11T, K15R, I17R, M18F, K19D, and
E32A, and is likely to be highly potent as a plasmin inhibitor.
DPI-1.1.2 carries D11T, K15R, I17R, M18F, and K19D, and is likely
to be highly potent. DPI-1.1.3 caries the mutations D11A, K15R,
I17R, M18F, and K19D relative to LACI-K1. DPI-1.1.3 differs from
DPI-1.1.2 by having A.sub.11 instead of T.sub.11; both proteins are
likely to be very potent plasmin inhibitors. DPI-1.1.4 carries the
mutations I17R, M18F, K19D, and E32A and should be quite potent. As
DPI-1.1.4 has fewer of the SPI11 mutations, it may be less potent,
but is also less likely to be immunogenic. DPI-1.1.5 carries the
mutations I17R, M18F, and K19D. This protein is likely to be a good
inhibitor and is less likely to be immunogenic. DPI-1.1.6 carries
only the mutations I17R and M18F but should inhibit plasmin.
[0069] Protein DPI-1.2.1 is based on human LACI-K2 and shown in
Table 4. The mutations P11T, I13P, Y17R, I18F, T19D, R32E, K34I,
and L39E are likely to confer high affinity for plasmin. Some of
these substitutions may not be necessary; in particular, P11T and
T19D may not be necessary. Other mutations that might improve the
plasmin affinity include E9A, D10E, G16A, Y21W, Y21F, R32T, K34V,
and L39G.
[0070] Protein DPI-1.3.1 (Table 4) is based on human LACI-K3. The
mutations R11T, L13P, N17R, E18F, N19D, R31E, P32E, K34I, and S36G
are intended to confer high affinity for plasmin. Some of these
substitutions may not be necessary; in particular, N19D and P32E
may not be necessary. Other changes that might improve K.sub.D
include D10E, N17K. F21W and G39E.
[0071] Protein DPI-2.1 (Table 4) is a based on the human collagen
.alpha.3 KuDom. The mutations E11T, T13P, D16A, F17R, I18F, L19D,
A3E, R32E, and W34I are likely to confer high affinity for plasmin.
Some of these substitutions may not be necessary; in particular,
L19D and A31E may not be necessary. Other mutations that might
improve the plasmin affinity include K9A, D10E, D16G, K20R, R32T,
W34V, and G39E.
[0072] DPI-3.1.1 (Table 4) is derived from Human TFPI-2 domain 1.
The exchanges Y11T, L17R, L18F, L19D, and R31E are likely to confer
high affinity for plasmin. The mutation L19D may not be needed.
Other mutations that might foster plasmin binding include Y21W,
Y21F, Q32E, L34I, L34V, and E39G.
[0073] DPI-3.2.1 (Table 4) is derived from Human TFPI-2 domain 2.
This parental domain contains insertions after residue 9 (one
residue) and 42 (two residues). The mutations (V.sub.9SVDDQC.sub.14
replaced by V.sub.9ETGPC.sub.14) E15R, S17K, T18F, K32T, F34V, and
(H.sub.39RNRIENR.sub.44 replaced by (E.sub.39GNRNR.sub.44) are
likely to confer affinity for plasmin. Because of the need to
change the number of amino acids, DPI-3.2.1 has a higher potential
for immunogenicity than do other modified human KuDoms.
[0074] DPI-3.3.1 (Table 4) is derived from human TFPI-2, domain 3.
The substitutions E11T, L13P, S15R, N17R, V18F, T34I, and T36G are
likely to confer high affinity for plasmin. The mutations E11T,
L13P, and T34I may not be necessary. Other mutations that might
foster plasmin binding include D10E, T19D, Y21W, and G39E.
[0075] DPI-4.1.1 (Table 4) is from human ITI-K1 by assertion of
S10E, M15R, M17K, T18F, Q34V, and M39G. The mutations M39G and Q34V
may not be necessary. Other mutations that should foster plasmin
binding include: A11T, G16A, M17R, S19D, Y21W, and Y21F.
[0076] DPI-4.2.1 (Table 4) is from human IT-K2 through the
mutations V10D, R11T, F17R, I18F, and P34V. The mutation P34V might
not be necessary. Other mutation that should foster plasmin binding
include: V10E, Q19D, L20R, W21F, P34I, and Q39E. DPI-4.2.2 is an
especially preferred protein as it has only three mutations: R11T,
F17R, and I18F. DPI-4.2.3 is an especially preferred protein as it
has only four mutations: R11T, F17R, I18F, and L20R. DPI-4.2.4 is
an especially preferred protein as it has only five mutations:
R11T, F17R, I18F, L20R, and P34V. DPI-4.2.5 carries the mutations
V10E, R11T, F17R, I18F, L20R, V31E, L32T, P34V, and Q39G and is
highly likely to inhibit plasmin very potently. Each of the
proteins DPI-4.2.1, DPI-4.2.2, DPI-4.2.3. DPI-4.2.4, and DPI-4.2.5
is very likely to be a highly potent inhibitor of plasmin.
[0077] Before DENN94a, it was thought that APP-I was a very potent
plasmin inhibitor. Thus, it was surprising to select proteins from
a library that was designed to allow the APP-I residues at
positions 10-21 which differed strongly from APP-I. Nevertheless,
APP-I can be converted into a potent plasmin inhibitor. DPI-5.1 is
derived from human APP-I (also known as Protease Nexin-II) by
mutations M17R and I18F and is likely to be a much better plasmin
inhibitor than is APP-I itself. DPI-5.2 carries the further
mutations S19D, A31E, and F34I which may foster higher affinity for
plasmin.
[0078] DPI-6.1 is derived from the HKI B9 KuDom (NORR93) by the
five substitutions: K11T, Q15R, T16A, M17R, and M18F. DPI-6.1 is
likely to be a potent plasmin inhibitor. DPI-6.2 carries the
additional mutations T19D and A34V which should foster plasmin
binding.
[0079] Although BPTI is the best naturally occurring KuDom plasmin
inhibitors known, it could be improved. DPI-7.1 is derived from
BPTI by the mutation I18F which is likely to increase the affinity
for plasmin. DPI-7.2 carries the further mutation K15R which should
increase plasmin binding. DPI-7.3 carries the added mutation R39G.
DPI-7.4 carries the mutations Y10D, K15R, I18F, I19D, Q31E, and
R39G and should have a very high affinity for plasmin.
Modification of Kunitz Domains
[0080] KuDoms are quite small; if this should cause a
pharmacological problem, such as excessively quick elimination from
circulation, two or more such domains may be joined. A preferred
linker is a sequence of one or more amino acids. A preferred linker
is one found between repeated domains of a human protein,
especially the linkers found in human BPTI homologues, one of which
has two domains (BALD85, ALBR83b) and another of which has three
(WUNT88). Peptide linkers have the advantage that the entire
protein may then be expressed by recombinant DNA techniques. It is
also possible to use a nonpeptidyl linker, such as one of those
commonly used to form immunogenic conjugates. An alternative means
of increasing the serum residence of a BPTI-like KuDom is to link
it to polyethyleneglycol, so called PEGylation (DAVI79).
Ways To Improve Specificity of SPI11 and Other KuDom Plasmin
Inhibitors:
[0081] Because we have made a large part of the surface of the
KuDom SPI11 complementary to the surface of plasmin, R.sub.15 is
not essential for specific binding to plasmin. Many of the enzymes
in the clotting and fibrinolylic pathways cut preferentially after
Arg or Lys. Not having a basic residue at the P1 position may give
rise to greater specificity. The variant SPI11-R15A (shown in Table
11), having an ALA at P1, is likely to be a good plasmin inhibitor
and may have higher specificity for plasmin relative to other
proteases than does SPI11. The affinity of SPI11-R15A for plasmin
is likely to be less than the affinity of SPI11 for plasmin, but
the loss of affinity for other Arg/Lys-preferring enzymes is likely
to be greater and, in many applications, specificity is more
important than affinity. Other mutants that are likely to have good
affinity and very high specificity include SPI11-R15G and
SPI11-R15N-E32A. This approach could be applied to other
high-affinity plasmin inhibitors.
Increasing the Affinity of SPI11
[0082] Variation of SPI11 as shown in Table 12 and selection of
binders is likely to produce a Kunitz domain having affinity for
plasmin that is higher than SPI11. This fourth library allows
variegation of the 14-38 disulfide. The two segments of DNA shown
are synthesized and used with primers in a PCR reaction to produce
ds DNA that runs from NsiI to BstEII. The primers are identical to
the 5' ends of the synthetic bits shown and of length 21 for the
first and 17 for the second. As the variability is very high, we
would endeavor to obtain between 10.sup.8 and 10.sup.9
transformants (the more the better).
Mode of Production
[0083] Proteins of this invention may be produced by any
conventional technique, including [0084] a) nonbiological synthesis
by sequential coupling of components, e.g. amino acids, [0085] b)
production by recombinant DNA techniques in suitable host cells,
and [0086] c) semisynthesis, for example, by removal of undesired
sequences from LACI-K1 and coupling of synthetic replacement
sequences.
[0087] Proteins disclosed herein are preferably produced,
recombinantly, in a suitable host, such as bacteria from the genera
Bacillus, Escherichia, Salmonella, Erwinia, and yeasts from the
genera Hansenula, Kluyveromyces, Pichia, Rhinosporidium,
Saccharomyces, and Schizosaccharomyces, or cultured mammalian cells
such as COS-1. The more preferred hosts are microorganisms of the
species Pichia pastoris, Bacillus subtilis, Bacillus brevis,
Saccharomyces cerevisiae, Escherichia coli and Yarrowia lipolytica.
Any promoter which is functional in the host cell may be used to
control gene expression.
[0088] Preferably the proteins are secreted and, most preferably,
are obtained from conditioned medium. Secretion is the preferred
route because proteins are more likely to fold correctly and can be
produced in conditioned medium with few contaminants. Secretion is
not required.
[0089] Unless there is a specific reason to include glycogroups, we
prefer proteins designed to lack N-liked glycosylation sites to
reduce potential for antigenicity of glycogroups and so that
equivalent proteins can be expressed in a wide variety of organisms
including: 1) E. coli, 2) B. subtilis, 3) P. pastoris, 4) S.
cerevisiae, and 5) mammalian cells.
[0090] Several means exist for reducing the problem of host cells
producing proteases that degrade the recombinant product; see,
infer alia BANE90 and BANE91. VAND92 reports that overexpression of
the B. subtilis signal peptidase in E. coli. leads to increased
expression of a heterologous fusion protein. ANBA88 reports that
addition of PMSF (a serine proteases inhibitor) to the culture
medium improved the yield of a fusion protein.
[0091] Other factors that may affect production of these and other
proteins disclosed here include: 1) codon usage (optimizing codons
for the host is preferred), 2) signal sequence, amino-acid sequence
at intended processing sites, presence and localization of
processing; enzymes, deletion, mutation, or inhibition of various
enzymes that might alter or degrade the engineered product and
mutations that make the host more permissive in secretion
(permissive secretion hosts are preferred).
[0092] Reference works on the general principles of recombinant DNA
technology include Watson et al., Molecular Biology of the Gene,
Volumes I and II, The Benjamin/Cummings Publishing Company, Inc.,
Menlo Park, Calif. (1987); Darnell et al., Molecular Cell Biology,
Scientific American Books, Inc., New York, N.Y. (1986); Lewin,
Genes II, John Wiley & Sons, New York, N.Y. (1985); Old, et
al., Principles of Gene Manipulation: An Introduction to Genetic
Engineering, 2d edition, University of California Press, Berkeley,
Calif. (1981); Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
(1989); and Ausubel et al, Current Protocols in Molecular Biology,
Wiley Interscience, NY, (1987, 1992). These references are herein
entirely incorporated by reference as are the references cited
therein.
Assays for Plasmin Binding and INHIBITION
[0093] Any suitable method may be used to test the compounds of
this invention. Scatchard (Ann NY Acad Sci (1949) 51:660-669)
described a classical method of measuring and analyzing binding
which is applicable to protein binding. This method requires
relatively pure protein and the ability to distinguish bound
protein from unbound.
[0094] A second appropriate method of measuring K.sub.D is to
measure the inhibitory activity against the enzyme. If the K.sub.D
to be measured is in the 1 nM to 1 .mu.M range, this method
requires chromogenic or fluorogenic substrates and lens of
micrograms to milligrams of relatively pure inhibitor. For the
proteins of this invention, having K.sub.D in the range 5 nM to 50
pM, nanograms to micrograms of inhibitor suffice. When using this
method, the competition between the inhibitor and the enzyme
substrate can give a measured K.sub.i that is higher than the true
K.sub.i. Measurement reported here are not so corrected because the
correction would be very small and the any correction would reduce
the K.sub.i. Here, we use the measured K.sub.i as a direct measure
of KD.
[0095] A third method of determining the affinity of a protein for
a second material is to have the protein displayed on a genetic
package, such as M13, and measure the ability of the protein to
adhere to the immobilized "second material". This method is highly
sensitive because the genetic packages can be amplified. We obtain
at least semiquantitative values for the binding constants by use
of a pH step gradient. Inhibitors of known affinity for the
protease are used to establish standard profiles against which
other phage-displayed inhibitors are judged. Any other suitable
method of measuring protein binding may be used.
[0096] Preferably, the proteins of this invention have a K.sub.D
for plasmin of at most about 5 nM, more preferably at most about
300 pM, and most preferably 100 pM or less. Preferably, the binding
is inhibitory so that K.sub.i is the same as K.sub.D. The K.sub.i
of QS4 for plasmin is about 2 nM. The K.sub.i of SPI11 for plasmin
is about 88 pM.
Pharmaceutical Methods and Preparations
[0097] The preferred subject of this invention is a mammal. The
invention is particularly useful in the treatment of humans, but is
suitable for veterinary applications too.
[0098] Herein, "protection" includes "prevention", "suppression",
and "treatment". "Prevention" involves administration of drug prior
to the induction of disease. "Suppression" involves administration
of drug prior to the clinical appearance of disease. "Treatment"
involves administration of drug after the appearance of
disease.
[0099] In human and veterinary medicine, it may not be possible to
distinguish between "preventing" and "suppressing" since the
inductive event(s) may be unknown or latent, or the patient is not
ascertained until after the occurrence of the inductive event(s).
We use the term "prophylaxis" as distinct from "treatment" to
encompass "preventing" and "suppressing". Herein, "protection"
includes "prophylaxis". Protection need not by absolute to be
useful.
[0100] Proteins of this invention may be administered, by any
means, systemically or topically, to protect a subject against a
disease or adverse condition. For example, administration of such a
composition may be by any parenteral route, by bolus injection or
by gradual perfusion. Alternatively, or concurrently,
administration may be by the oral route. A suitable regimen
comprises administration of an effective amount of the protein,
administered as a single dose or as several doses over a period of
hours, days, months, or years.
[0101] The suitable dosage of a protein of this invention may
depend on the age, sex, health, and weight of the recipient, kind
of concurrent treatment, if any, frequency of treatment, and the
desired effect. However, the most preferred dosage can be tailored
to the individual subject, as is understood and determinable by one
of skill in the art, without undue experimentation by adjustment of
the dose in ways known in the art.
[0102] For methods of preclinical and clinical testing of drugs,
including proteins, see, e.g., Berkow et al, eds., The Merck
Manual, 15th edition, Merck and Co., Rahway, N.J., 1987; Goodman et
al., eds., Goodman and Gilman's The Pharmacological Basis of
Therapeutics, 8th edition, Pergamon Press, Inc., Elmsford, N.Y.,
(1990); Avery's Drug Treatment: Principles and Practice of Clinical
Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD.,
Williams and Wilkins, Baltimore, Md. (1987), Ebadi, Pharmacology,
Little, Brown and Co., Boston, (1985), which references and
references cited there are hereby incorporated by reference.
[0103] In addition to a protein here disclosed, a pharmaceutical
composition may contain pharmaceutically acceptable carriers,
excipients, or auxiliaries. See, e.g., Berker, supra, Goodman,
supra, Avery, supra and Ebadi, supra.
In Vitro Diagnostic Methods and Reagents
[0104] Proteins of this invention may be applied in vitro to any
suitable sample that might contain plasmin to measure the plasmin
present. To do so, the assay must include a Signal Producing System
(SPS) providing a detectable signal that depends on the amount of
plasmin present. The signal may be detected visually or
instrumentally. Possible signals include production of colored,
fluorescent, or luminescent products, alteration of the
characteristics of absorption or emission of radiation by an assay
component or product and precipitation or agglutination of a
component or product.
[0105] The component of the SPS most intimately associated with the
diagnostic reagent is called the "label". A label may be, e.g., a
radioisotope, a fluorophore, an enzyme, a co-enzyme, an enzyme
substrate, an electron-dense compound, or an agglutinable particle.
A radioactive isotope can be detected by use of, for example, a
.gamma. counter or a scintillation counter or by autoradiography.
Isotopes which are particularly useful are .sup.3H, .sup.125I,
.sup.131I, .sup.35S, .sup.14C, and, preferably, .sup.125I. It is
also possible to label a compound with a fluorescent compound. When
the fluorescently labeled compound is exposed to light of the
proper wave length, its presence can be detected. Among the most
commonly used fluorescent labelling compounds are fluorescein
isothiocyanate, rhodamine, phycoerythrin, phycocyanin,
allophycocyanin, o-phthaldehyde, and fluorescamine. Alternatively,
fluorescence-emitting metals, such as .sup.125Eu or other
lanthanide, may be attached to the binding protein using such metal
chelating groups as diethylenetriaminepentaacetic acid or
ethylenediamine-tetraacetic acid. The proteins also can be
detectably labeled by coupling to a chemiluminescent compound, such
as luminol, isolumino, theromatic acridinium ester, imidazole,
acridinium salt, and oxalate ester. Likewise, a bioluminescent
compound, such as luciferin, luciferase and aequorin, may be used
to label the binding protein. The presence of a bioluminescent
protein is determined by detecting the presence of luminescence.
Enzyme labels, such as horseradish peroxidase and alkaline
phosphatase, are preferred.
[0106] There are two basic types of assays: heterogeneous and
homogeneous. In heterogeneous assays, binding of the affinity
molecule to analyte does not affect the label; thus, to determine
the amount of analyte, bound label must be separated from free
label. In homogeneous assays, the interaction does affect the
activity of the label, and analyte can be measured without
separation.
[0107] In general, a plasmin-binding protein (PBP) may be used
diagnostically in the same way that an antiplasmin antibody is
used. Thus, depending on the assay format, it may be used to assay
plasmin, or, by competitive inhibition, other substances which bind
plasmin.
[0108] The sample will normally be a biological fluid, such as
blood, urine, lymph, semen, milk, or cerebrospinal fluid, or a
derivative thereof, or a biological tissue, e.g., a tissue section
or homogenate. The sample could be anything. If the sample is a
biological fluid or tissue, it may be taken from a human or other
mammal, vertebrate or animal, or from a plant. The preferred sample
is blood, or a fraction or derivative thereof.
[0109] In one embodiment, the plasmin-binding protein (PBP) is
immobilized, and plasmin in the sample is allowed to compete with a
known quantity of a labeled or specifically labelable plasmin
analogue. The "plasmin analogue" is a molecule capable of competing
with plasmin for binding to the PBP, which includes plasmin itself.
It may be labeled already, or it may be labeled subsequently by
specifically binding the label to a moiety differentiating the
plasmin analogue from plasmin. The phases arc separated, and the
labeled plasmin analogue in one phase is quantified.
[0110] In a "sandwich assay", both an insolubilized plasmin-binding
agent (PBA), and a labeled PBA are employed. The plasmin analyte is
captured by the insolubilized PBA and is tagged by the labeled PBA,
forming a tertiary complex. The reagents may be added to the sample
in any order. The PBAs may be the same or different, and only one
PBA need be a PBP according to this invention (the other may be,
e.g., an antibody). The amount of labeled PBA in the tertiary
complex is directly proportional to the amount of plasmin in the
sample.
[0111] The two embodiments described above are both heterogeneous
assays. A homogeneous assay requires only that the label be
affected by the binding of the PBP to plasmin. The plasmin analyte
may act as its own label if a plasmin inhibitor is used as a
diagnostic reagent.
[0112] A label may be conjugated, directly or indirectly (e.g.,
through a labeled anti-PBP antibody), covalently (e.g., with SPDP)
or noncovalently, to the plasmin-binding protein, to produce a
diagnostic reagent. Similarly, the plasmin binding protein may be
conjugated to a solid phase support to form a solid phase
("capture") diagnostic reagent. Suitable supports include glass,
polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,
and magnetite. The carrier can be soluble to some extent or
insoluble for the purposes of this invention. The support material
may have any structure so long as the coupled molecule is capable
of binding plasmin.
In Vivo Diagnostic Uses
[0113] A Kunitz domain that binds very tightly to plasmin can be
used for in vivo imaging. Diagnostic imaging of disease foci was
considered one of the largest commercial opportunities for
monoclonal antibodies, but this opportunity has not been achieved.
Despite considerable effort, only two monoclonal antibody-based
imaging agents have been approved. The disappointing results
obtained with monoclonal antibodies is due in large measure to:
[0114] i) Inadequate affinity and/or specificity; [0115] ii) Poor
penetration to target sites; [0116] iii) Slow clearance from
nontarget sites; [0117] iv) Immunogenicity (most are murine); and
[0118] v) High production cost and poor stability.
[0119] These limitations have led most in the diagnostic imaging
field to begin to develop peptide-based imaging agents. While
potentially solving the problems of poor penetration and slow
clearance, peptide-based imaging agents are unlikely to possess
adequate affinity, specificity and in vivo stability to be useful
in most applications.
[0120] Engineered proteins are uniquely suited to the requirements
for an imaging agent. In particular the extraordinary affinity and
specificity that is obtainable by engineering small, stable,
human-origin protein domains having known its vivo clearance rates
and mechanisms combine to provide earlier, more reliable results,
less toxicity/side effects, lower production and storage cost, and
greater convenience of label preparation. Indeed, it should be
possible to achieve the goal of realtime imaging with engineered
protein imaging agents. Plasmin-binding proteins, e.g. SPI11, may
be useful for localizing sites of internal hemorrhage.
[0121] Radio-labelled binding protein may be administered to the
human or animal subject. Administration is typically by injection,
e.g., intravenous or arterial or other means of administration in a
quantity sufficient to permit subsequent dynamic and/or static
imaging using suitable radio-detecting devices. The dosage is the
smallest amount capable of providing a diagnostically effective
image, and may be determined by means conventional in the art,
using known radio-imaging agents as guides.
[0122] Typically, the imaging is carried out on the whole body of
the subject, or on that portion of the body or organ relevant to
the condition or disease under study. The radio-labelled binding
protein has accumulated. The amount of radio-labelled binding
protein accumulated at a given point in time in relevant target
organs can then be quantified.
[0123] A particularly suitable radio-detecting device is a
scintillation camera, such as a .gamma. camera. The detection
device in the camera senses and records (and optional digitizes)
the radioactive decay. Digitized information can be analyzed in any
suitable way, many of which are known in the art. For example, a
time-activity analysis can illustrate uptake through clearance of
the radio-labelled binding protein by the target organs with
time.
[0124] Various factors are taken into consideration in picking an
appropriate radioisotope. The isotope is picked: to allow good
quality resolution upon imaging, to be safe for diagnostic use in
humans and animals, and, preferably, to have a short half-life so
as to decrease the amount of radiation received by the body. The
radioisotope used should preferably be pharmacologically inert, and
the quantities administered should not have substantial
physiological effect. The binding protein may be radio-labelled
with different isotopes of iodine, for example .sup.123I,
.sup.125I, or .sup.131I (see, for example, U.S. Pat. No.
4,609,725). The amount of labeling must be suitably monitored.
[0125] In applications to human subjects, it may be desirable to
use radioisotopes other than .sup.125I for labelling to decrease
the total dosimetry exposure of the body and to optimize the
detectability of the labeled molecule. Considering ready clinical
availability for use in humans, preferred radio-labels include:
.sup.99mTc, .sup.67Ga, .sup.68Ga, .sup.90Y, .sup.111In,
.sup.113mIn, .sup.123I, .sup.186Re, .sup.188Re or .sup.211At.
Radio-labelled protein may be prepared by various methods. These
include radio-halogenation by the chloramine-T or lactoperoxidase
method and subsequent purification by high pressure liquid
chromatography, for example, see Gutkowska et al in "Endocrinology
and Metabolism Clinics of America: (1987) 16 (1):183. Other methods
of radio-labelling can be used, such as IODOBEADS.TM..
[0126] A radio-labelled protein may be administered by any means
that enables the active agent to reach the agent's site of action
in a mammal. Because proteins are subject to digestion when
administered orally, parenteral administration, i.e., intravenous
subcutaneous, intramuscular, would ordinarily be used to optimize
absorption.
Other Uses
[0127] The plasmin-binding proteins of this invention may also be
used to purify plasmin from a fluid, e.g., blood. For this purpose,
the PBP is preferably immobilized on an insoluble support. Such
supports include those already mentioned as useful in preparing
solid phase diagnostic reagents.
[0128] Proteins can be used as molecular weight markers for
reference in the separation or purification of proteins. Proteins
may need to be denatured to serve as molecular weight markers. A
second general utility for proteins is the use of hydrolyzed
protein as a nutrient source. Proteins may also be used to increase
the viscosity of a solution.
[0129] The protein of this invention may be used for any of the
foregoing purposes, as well as for therapeutic and diagnostic
purposes as discussed further earlier in this specification.
Preparation of Peptides
[0130] Chemical polypeptide synthesis is a rapidly evolving area in
the art, and methods of solid phase polypeptide synthesis are
well-described in the following references, hereby entirely
incorporated by reference: (Merrifield, J Amer Chem Soc
85:2149-2154 (1963); Merrifield, Science 232:341-347 (1986); Wade
et al., Biopolymers 25:S21-S37 (1986); Fields, Int J Polypeptide
Prot Res 35:161 (1990); MilliGen Report Nos. 2 and 2a, Millipore
Corporation, Bedford, Mass., 1987) Ausubel et al, supra, and
Sambrook et al, supra. Tan and Kaiser (Biochemistry, 1977,
16:1531-41) synthesized BPTI and a homologue eighteen years
ago.
[0131] As is known in the art, such methods involve blocking or
protecting reactive functional groups, such as free amino, carboxyl
and thio groups. After polypeptide bond formation, the protective
groups are removed. Thus, the addition of each amino acid residue
requires several reaction steps for protecting and deprotecting.
Current methods utilize solid phase synthesis, wherein the
C-terminal amino acid is covalently linked to an insoluble resin
particles that can be filtered. Reactants are removed by washing
the resin particles with appropriate solvents using an automated
machine. Various methods, including the "tBoc" method and the
"Fmoc" method are well known in the art. See, inter alia, Atherton
et al., J Chem Soc Perkin Trans 1:538-546 (1981) and Sheppard et
al., Int J Polypeptide Prot Res 20:451-454 (1982).
EXAMPLES
Example 1
Construction of LACI (K1) Library
[0132] A synthetic oligonucleotide duplex having NsiI- and
MluI-compatible ends was cloned into a parental vector
(LACI-K1::III) previously cleaved with the above two enzymes. The
resultant ligated material was transfected by electroporation into
XLIMR (F.sup.-) E. coli strain and plated on ampicillin (Ap) plates
to obtain phage-generating Ap.sup.R colonies. The variegation
scheme for Phase 1 focuses on the P1 region, and affected residues
13, 16, 17, 18 and 19. It allowed for 6.6.times.10.sup.5 different
DNA sequences (3.1.times.10.sup.5 different protein sequences). The
library obtained consisted of 1.4.times.10.sup.6 independent cfu's
which is approximately a two fold representation of the whole
library. The phage stock generated from this plating gave a total
titer of 1.4.times.10.sup.13 pfu's in about 3.9 ml, with each
independent done being represented, on average, 1.times.10.sup.7 in
total and 2.6.times.10.sup.6 times per ml of phage stock.
[0133] To allow for variegation of residues 31, 32, 34 and 39
(phase II), synthetic oligonucleotide duplexes with MluI- and
BstEII-compatible ends were cloned into previously cleaved R.sub.f
DNA derived from one of the following [0134] i) the parental
construction, [0135] ii) the phase I library, or [0136] iii)
display phage selected from the first phase binding to a given
target.
[0137] The variegation scheme for phase II allows for 4096
different DNA sequences (1600 different protein sequences) due to
alterations at residues 31, 32, 34 and 39. The final phase II
variegation is dependent upon the level of variegation remaining
following the three rounds of binding and elution with a given
target in phase I.
[0138] The combined possible variegation for both phases equals
2.7.times.10.sup.8 different DNA sequences or 5.0.times.10.sup.7
different protein sequences. When previously selected display phage
are used as the origin of R.sub.f DNA for the phase II variegation,
the final level of variegation is probably in the range of 10.sup.5
to 10.sup.6.
Example 2
Screening of LACI-K1 Library for Binding to Plasmin
[0139] The scheme for selecting LACI-K1 variants that bind plasmin
involves incubation of the phage-display library with plasmin-beads
(Calbiochem, San Diego, Calif.; catalogue no. 527802) in a buffer
(PBS containing 1 mg/ml BSA) before washing away unbound and poorly
retained display-phage variants with PBS containing 0.1% Tween 20.
The more strongly bound display-phage are eluted with a low pH
elution buffer, typically citrate buffer (pH 2.0) containing 1
mg/ml BSA, which is immediately neutralized with Tris buffer to pH
7.5. This process constitutes a single round of selection.
[0140] The neutralized eluted display-phage can be either used:
[0141] i) to inoculate an F.sup.+ strain of E. coli to generate a
new display-phage stock, to be used for subsequent rounds of
selection (so-called conventional screening), or [0142] ii) be used
directly for another immediate round of selection with the protease
beads (so-called quick screening).
[0143] Typically, three rounds of either method, or a combination
of the two, are performed to give rise to the final selected
display-phage from which a representative number are sequenced and
analyzed for binding properties either as pools of display-phage or
as individual clones.
[0144] For the LACI-K1 library, two phases of selection were
performed, each consisting of three rounds of binding and elution.
Phase I selection used the phase I library (variegated residues 13,
16, 17, 18, and 19) which went through three rounds of binding and
elution against plasmin giving rise to a subpopulation of clones.
The R.sub.f DNA derived from this selected subpopulation was used
to generate the Phase II library (addition of variegated residues
31, 32, 34 and 39). About 5.6.times.10.sup.7 independent
transformants were obtained. The phase II libraries underwent three
further rounds of binding and elution with the same target protease
giving rise to the final selectants.
[0145] Following two phases of selection against plasmin-agarose
beads a representative number (16) of final selection display-phage
were sequenced. Table 2 shows the sequences of the selected LACI-K1
domains with the amino acids selected at the variegated positions
in upper case. Note the absolute selection of residues P.sub.13,
A.sub.16, R.sub.17, F.sub.18, and E.sub.19. There is very strong
selection for E at 31 and Q at 32. There is no consensus at 34; the
observed amino acids are (T.sub.3, Y.sub.2, H.sub.2, D, R, A,
V.sub.2, I.sub.3, and L). The amino acids having side groups that
branch at C.sub.p (T, I, and V) are multiply represented and are
preferred. At position 39, there is no strong consensus (G.sub.6,
D.sub.3, Q.sub.2, A.sub.2, R, F, E), but G, D, Q, and A seem to be
preferred (in that order).
[0146] A separate screening of the LACI-K1 library against plasmin
gave a very similar consensus from 16 sequenced selected
display-phage. These sequences are shown in Table 3 (selected
residues in upper case). These sequences depart from those of Table
2 in that E here predominates at position 19. There is a consensus
at 34 (T.sub.5, V.sub.3, S.sub.3, I.sub.2, L, A, F) of T. V, or S.
Combining the two sets, there is a preference for (in order of
preference) T, V, I, S, A, H, Y, and L, with F, D, and R being
allowed.
Expression, Purification and Kinetic Analysis.
[0147] The three isolates QS4, ARFK#1, and ARFK#2 were recloned
into a yeast expression vector. The yeast expression vector is
derived from pMFalpha8 (KURJ82 and MIYA85). The LACI variant genes
were fused to part of the mat.alpha.1 gene, generating a hybrid
gene consisting of the mat.alpha.1 promoter-signal peptide and
leader sequence-fused to the LACI variant. The cloning site is
shown in Table 24. Note that the correctly processed LACI-K1
variant protein should be as detailed in Table 2 and Table 3 with
the addition of residues glu-ala-ala-glu to the N-terminal met
(residue 1 in Table 2 and Table 3). Expression in S. cerevisiae
gave a yield of about 500 .mu.g of protease inhibitor per liter of
medium. Yeast-expressed LACI (kunitz domain 1), BPTI and LACI
variants: QS4, ARFK#1 and ARFK#2 were purified by affinity
chromatography using trypsin-agarose beads.
[0148] The most preferred production host is Pichia pastoris
utilizing the alcohol oxidase system Other have produced a number
of proteins in the yeast Pichia pastoris. For example, Vedvick et
al. (VEDV91) and Wagner et al. (WAGN92) produced aprotinin from the
alcohol oxidase promoter with induction by methanol as a secreted
protein in the culture medium at .apprxeq.1 mg/ml. Gregg et al.
(GREG93) have reviewed production of a number of proteins in P.
pastoris. Table 1 of GREG93 shows proteins that have been produced
in P. pastoris and the yields.
Kinetic Data
[0149] Inhibition of hydrolysis of
succinyl-Ala-Phe-Lys-(F.sub.3Ac)AMC (a methyl coumarin) (Sigma
Chemical, St. Louis, Mo.) by plasmin at 2.5.times.10.sup.-8 M with
varying amount of inhibitor were fit to the standard form for a
tight-binding substrate by least-squares. Preliminary kinetic
analysis of the two ARFK variants demonstrated very similar
inhibitory activity to that of the QS4 variant.) These measurements
were carried out with physiological amounts of salt (150 mM) so
that the affinities are relevant to the action of the proteins in
blood.
[0150] Table 23 shows that QS4 is a highly specific inhibitor of
human plasmin. Phage that display the LACI-K1 derivative QS4 bind
to plasmin beads at least 50-times more than it binds to other
protease targets.
New Library for Plasmin:
[0151] A new library of LACI-K1 domains, displayed on M13 gIIIp and
containing the diversity shown in Table 5 was made and screened for
plasmin binding. Table 6 shows the sequences selected and the
consensus. We characterized the binding of the selected proteins by
comparing the binding of clonally pure phage to BPTI display phage.
Isolates 11, 15, 08, 23, and 22 were superior to BPTI phage. We
produced soluble SPI11 (Selected Plasmin Inhibitor#11) and tested
its inhibitory activity, obtaining a K.sub.i of 88 pM which is at
least two-fold better than BPTI. Thus, we believe that the
selectants SPI15, SPI08, and SPI22 are far superior to BPTI and
that SPI23 is likely to be about as potent as BPTI. AU of the
listed proteins are much closer to a human protein amino-acid
sequence than is BPTI and so have less potential for
immunogenicity.
[0152] All references, including those to U.S. and foreign patents
or patent applications, and to nonpatent disclosures, are hereby
incorporated by reference in their entirety.
TABLE-US-00002 TABLE 1 Sequence of whole LACI: (SEQ ID NO. 1) 5 5 5
5 5 1 MIYTMKKVHA LWASVCLLLN LAPAPLNAds eedeehtiit dtelpplklM 51
HSFCAFKADD GPCKAIMKRF FFNIFTRQCE EFIYGGCEGN QNRFESLEEC 101
KKMCTRDnan riikttlqqe kpdfCfleed pgiCrgyitr yfynnqtkqC 151
erfkyggClg nmnnfetlee CkniCedgpn gfqvdnygtq lnavnnsltp 201
qstkvpslfe fhgpswCltp adrglCrane nrfyynsvig kCrpfkysgC 251
ggnennftsk qeClraCkkg fiqriskggl iktkrkrkkq rvkiayeeif 301 vknm
[0153] The signal sequence (1-28) is uppercase and underscored
[0154] LACI-K1 is uppercase
[0155] LACI-K2 is underscored
[0156] LACI-K3 is bold
TABLE-US-00003 TABLE 2 Sequence of CLACI-K1 and derivatives that
bind human plasmin 1 2 3 4 5
1234567890123456789012345678901234567890123456789012345678 LACI-K1
mhsfcafkaddgpckaimkrfffniftrqceefiyggcegnqnrfesleeckkmctrd SEQ ID
NO. 2 QS1
mhsfcafkaddgPckARFErfffniftrqcEQfTyggcRgnqnrfesleeckkmctrd SEQ ID
NO. 3 QS4
mhsfcafkaddgPckARFErfffniftrqcEQfYyggcDgnqnrfesleeckkmctrd SEQ ID
NO. 4 QS7
mhsfcafkaddgPckARFErfffniftrqcEQfHyggcDgnqnrfesleeckkmctrd SEQ ID
NO. 5 QS8
mhsfcafkaddgPckARFErfffniftrqcEQfDyggcAgnqnrfesleeckkmctrd SEQ ID
NO. 6 QS9
mhsfcafkaddgPckARFErfffniftrqcQEfRyggcDgnqnrfesleeckkmctrd SEQ ID
NO. 7 QS13
mhsfcafkaddgPckARFErfffniftrqcQQfYyggcQgnqnrfesleeckkmctrd SEQ ID
NO. 8 QS15
mhsfcafkaddgPckARFErfffniftrqcEEfAyggcGgnqnrfesleeckkmctrd SEQ ID
NO. 9 NS2
mhsfcafkaddgPckARFErfffniftrqcQQfVyggcGgnqnrfesleeckkmctrd SEQ ID
NO. 10 NS4
mhsfcafkaddgPckARFErfffniftrqcEQfTyggcGgnqnrfesleeckkmctrd SEQ ID
NO. 11 NS6
mhsfcafkaddgPckARFErfffniftrqcEEfTyggcGgnqnrfesleeckkmctrd SEQ ID
NO. 12 NS9
mhsfcafkaddgPckARFErfffniftrqcEQfIyggcQgnqnrfesleeckkmctrd SEQ ID
NO. 13 NS11
mhsfcafkaddgPckARFErfffniftrqcEQfIyggcGgnqnrfesleeckkmctrd SEQ ID
NO. 14 NS12
mhsfcafkaddgPckARFErfffniftrqcEQfIyggcFgnqnrfesleeckkmctrd SEQ ID
NO. 15 NS14
mhsfcafkaddgPckARFErfffniftrqcQQfHyggcEgnqnrfesleeckkmctrd SEQ ID
NO. 16 NS15
mhsfcafkaddgPckARFErfffniftrqcEQfVyggcAgnqnrfesleeckkmctrd SEQ ID
NO. 17 NS16
mhsfcafkaddgPckARFErfffniftrqcEQfLyggcGgnqnrfesleeckkmctrd SEQ ID
NO. 18 ARFRCON
mhsfcafkaddgPckARFErfffniftrqcEQfiyggcGgnqnrfesleeckkmctrd SEQ ID
NO. 19
TABLE-US-00004 TABLE 3 Sequence of LACI-K1 and derivatives that
bind human plasmin 1 2 3 4 5
1234567890123456789012345678901234567890123456789012345678 LACI-K1
mhsfcafkaddgpckaimkrfffniftrqceefiyggcegnqnrfesleeckkmctrd SEQ ID
NO. 2 ARFK#1
mhsfcafkaddgPckARFErfffniftrqcEQfVyggcGgnqnrfesleeckkmctrd SEQ ID
NO. 20 ARFK#2
mhsfcafkaddgPckARFErfffniftrqcEEfVyggcGgnqnrfesleeckkmctrd SEQ ID
NO. 21 ARFK#3
mhsfcafkaddgLckGRFQrfffniftrqcEEfIyggcEgnqnrfesleeckkmctrd SEQ ID
NO. 22 ARFK#4
mhsfcafkaddgPckARFErfffniftrqcEQfTyggcMgnqnrfesleeckkmctrd SEQ ID
NO. 23 ARFK#5
mhsfcafkaddgPckARFErfffniftrqcEQfSyggcGgnqnrfesleeckkmctrd SEQ ID
NO. 24 ARFK#6
mhsfcafkaddgPckARFErfffniftrqcEEfLyggcLgnqnrfesleeckkmctrd SEQ ID
NO. 25 ARFK#7
mhsfcafkaddgPckARFErfffniftrqcEQfSyggcQgnqnrfesleeckkmctrd SEQ ID
NO. 26 ARFK#8
mhsfcafkaddgPckARFErfffniftrqcEQfAyggcAgnqnrfesleeckkmctrd SEQ ID
NO. 27 ARFK#9
mhsfcafkaddgPckARFErfffniftrqcEQfIyggcVgnqnrfesleeckkmctrd SEQ ID
NO. 28 ARFK#10
mhsfcafkaddgPckARFErfffniftrqcEEfSyggcKgnqnrfesleeckkmctrd SEQ ID
NO. 29 ARFK#11
mhsfcafkaddgPckARFErfffniftrqcEEfVyggcKgnqnrfesleeckkmctrd SEQ ID
NO. 30 ARFK#12
mhsfcafkaddgPckASFErfffniftrqcEQfTyggcNgnqnrfesleeckkmctrd SEQ ID
N0. 31 ARFK#13
mhsfcafkaddgPckASFErfffniftrqcEEfTyggcLgnqnrfesleeckkmctrd SEQ ID
NO. 32 ARFK#14
mhsfcafkaddgPckARFErfffniftrqcEQfFyggcHgnqnrfesleeckkmctrd SEQ ID
NO. 33 ARFK#15
mhsfcafkaddgPckARFErfffniftrqcEQfTyggcGgnqnrfesleeckkmctrd SEQ ID
NO. 34 ARFK#16
mhsfcafkaddgPckARFErfffniftrqcEQfTyggcMgnqnrfesleeckkmctrd SEQ ID
NO. 35 ARFKC01
mhsfcafkaddgPckARFErfffniftrqcEQfTyggcGgnqnrfesleeckkmctrd SEQ ID
NO. 36 ARFKC02
mhsfcafkaddgPckARFErfffniftrqcEQfVyggcGgnqnrfesleeckkmctrd SEQ ID
NO. 37
TABLE-US-00005 TABLE 4 Kunitz domains, some of which inhibit
plasmin Amino-acid Sequence Affin- Protein
111111111122222222223333333333444444444A555555555 ity identifier
1234567890123456789012345678901234567890123456789012345678 K.sub.D
SEQ ID NO. QS4
mhsfcafkaddgPckARFErfffniftrqcEQfYyggcDgnqnrfesleeckkmctrd 2 nM SEQ
ID NO. 4 NS4
mhsfcafkaddgPckARFErfffniftrqcEQfTyggcGgnqnrfesleeckkmctrd (B) SEQ
ID NO. 11 BPTI
RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGA .3 nM
SEQ ID NO. 38 Human
VREVCSEQAETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSA 75 pM
SEQ ID NO. 39 APP-I (KIDO88) 225 nM (DENN94a) SPI11
mhsfcafkaETgPcRARFDrWffniftrqceefiyggcegnqnrfesleeckkmctrd 88 pM
SEQ ID NO. 40 SPI15
mhsfcafkaESgPcRARFDrWffniftrqceefiyggcegnqnrfesleeckkmctrd (A) SEQ
ID NO. 41 SPI08
mhsfcafkaDGgPcRARFErFffniftrqceefiyggcegnqnrfesleeckkmctrd (A) SEQ
ID NO. 42 SPI23
mhsfcafkaEGgPcRAKFQrWffniftrqceefiyggcegnqnrfesleeckkmctrd ~.5 nM
SEQ ID NO. 43 SPI22
mhsfcafkaDGgPcKGKFPrFffniftrqceefiyggcegnqnrfesleeckkmctrd >2 nM
SEQ ID NO. 44 SPIcon1
mhsfcafkaETgPcRAkFDrWffniftrqcEAfVyggcGgnqnrfesleeckkmctrd SEQ ID
NO. 45 SPI60
mhsfcafkaETgPcRAkFDrWffniftrqcEPfVYggcEgnqnrfesleeckkmctrd (B) SEQ
ID NO. 46 SPI59
mhsfcafkaETgPcRAkFDrWffniftrqcNTfVYggcGgnqnrfesleeckkmctrd SEQ ID
NO. 47 SPI42
mhsfcafkaETgPcRGkFDrWffniftrqcQGfVYggcGgnqnrfesleeckkmctrd SEQ ID
NO. 48 SPI55
mhsfcafkaEVgPcRAkFDrWffniftrqcHLfTYggcGgnqnrfesleeckkmctrd SEQ ID
NO. 49 SPI56
mhsfcafkaETgPcRGkFDrWffniftrqcAQfVYggcEgnqnrfesleeckkmctrd SEQ ID
NO. 50 SPI43
mhsfcafkaETgPcRGkFDrWffniftrqcESfHYggcKgnqnrfesleeckkmctrd >-4
nM SEQ ID NO. 51 SPI52
mhsfcafkaDAgPcRAkFErFffniftrqcEAfLYggcGgnqnrfesleeckkmctrd SEQ ID
NO. 52 SPI46
mhsfcafkaDVgPcRAkFErFffniftrqcEAfLYggcEgnqnrfesleeckkmctrd SEQ ID
NO. 53 SPI51
mhsfcafkaDAgPcRAkFErFffniftrqcTAfFYggcGgnqnrfesleeckkmctrd ~.5 nM
SEQ ID NO. 54 SPI54
mhsfcafkaDSgPcRARFDrWffniftrqcTRfPYggcGgnqnrfesleeckkmctrd >-4
nM SEQ ID NO. 55 SPI49
mhsfcafkaETgPcRAkIPrLffniftrqcEPfIWggcGgnqnrfesleeckkmctrd SEQ ID
NO. 56 SPI47
mhsfcafkaDAgPcRAkFErFffniftrqcEEfIYggcEgnqnrfesleeckkmctrd ~.8 nM
SEQ ID NO. 57 SPI53
mhsfcafkaETgPcKGSFDrWffniftrqcNVfRyggcRgnqnrfesleeckkmctrd SEQ ID
NO. 58 SPI41
mhsfcafkaDAgPcRARFErFffniftrqcDTfLYggcEgnqnrfesleeckkmctrd (AB) SEQ
ID NO. 59 SPI57
mhsfcafkaDSgPcKGRFGrLffniftrqcTAfDWggcGgnqnrfesleeckkmctrd SEQ ID
NO. 60 DPI-1.1.1
mhsfcafkadTgpcRaRFDrfffniftrqceAfiyggcegnqnrfesleeckkmctrd (A) SEQ
ID NO. 61 DPI-1.1.2
mhsfcafkadTgpcRaRFDrfffniftrqceefiyggcegnqnrfesleeckkmctrd (A) SEQ
ID NO. 62 DPI-1.1.3
mhsfcafkadAgpcRaRFDrfffniftrqceefiyggcegnqnrfesleeckkmctrd (A) SEQ
ID NO. 63 DPI-1.1.4
mhsfcafkadTgpckaRFDrfffniftrqceAfiyggcegnqnrfesleeckkmctrd (AB) SEQ
ID NO. 127 DPI-1.1.5
mhsfcafkaddgpckaRFDrfffniftrqceefiyggcegnqnrfesleeckkmctrd (B) SEQ
ID NO. 128 DPI-1.1.6
mhsfcafkaddgpckaRFkrfffniftrqceefiyggcegnqnrfesleeckkmctrd (C) SEQ
ID NO. 129 Human
KPDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLEECKNICEDG SEQ ID
NO. 64 LACI-K2 DPI-1.2.1
kpdfcfleedTgPcrgRFDryfynngtkqceTfIyggcEgnmnnfetleecknicedg SEQ ID
NO. 65 Human
GPSWCLTPADRGLCRANENRFYYNSVIGKCRPFKYSGCGGNENNFTSKQECLRACKKG SEQ ID
NO. 66 LACI-K3 DPI-1.3.1
gpswcltpadTgPcraRFDrfyynsvigkcEpfIyGgcggnennftskqeclrackkg SEQ ID
NO. 67 Human
ETDICKLPKDEGTCRDFILKWYYDPNTKSCARFWYGGCGGNENKFGSQKECEKVCAPV SEQ ID
NO. 68 collagen .alpha.3 KuDom DPI-2.1
etdicklpkdTgPcrARFDkwyydpntkscEPfVyggcggnenkfgsqkecekvcapv SEQ ID
NO. 69 Human
NAEICLLPLDYGPCRALLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRI SEQ ID
NO. 70 TFPI-2 DOMAIN 1 DPI-3.1.1
naeicllpldTgpcraRFDryyydrytqscEqflyggcegnannfytweacddacwri SEQ ID
NO. 71 Human VPKVCRLQV- SEQ ID NO. 72 TFPI-2
SVDDQCEGSTEKYFFNLSSMTCEKFFSGGCHRNR- DOMAIN 2 IENRFPDEATCMGFCAPK
DPI-3.2.1
vpkvcrlqvETGPcRgKFekyffnlssmtceTfVYggcEGnrnrfpdeatcmgfcapk SEQ ID
NO. 73 Human
IPSFCYSPKDEGLCSANVTRYYFNPRYRTCDAFTYTGCGGNDNNFVSREDCKRACAKA SEQ ID
NO. 74 TFPI-2 DOMAIN 3 DPI-3.3.1
ipsfcyspkdTgPcRaRFtryyfnpryrtcdaftyGgcggndnnfvsredckracaka SEQ ID
NO. 75 HUMAN
KEDSCQLGYSAGPCMGMTSRYFYNGTSMACETFQYGGCMGNGNNFVTEKECLQTCRTV SEQ ID
NO. 76 ITI-K1 DPI-4.1.1
kedscqlgyEagpcRgKFsryfyngtsmacetfVyggcGgngnnfvtekeclqtcrtv SEQ ID
NO. 77 Human
TVAACNLPIVRGPCRAFIQLWAFDAVKGKCVLFPYGGCQGNGNKFYSEKECREYCGVP SEQ ID
NO. 78 ITI-K2 DPI-4.2.1
tvaacnlpiDTgpcraRFqlwafdavkgkcvlfVyggcqgngnkfysekecreycgvp SEQ ID
NO. 79 PROTEASE
VREVCSEQAETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSA SEQ ID
NO. 80 NEXIN-II DPI-5.1
vrevcseqaetgpcraRFsrwyfdvtegkcapffyggcggnrnnfdteeycmavcgsa SEQ ID
NO. 81 DPI-5.2
vrevcseqaetgpcraRFsrwyfdvtegkcEpfIyggcggnrnnfdteeycmavcgsa SEQ ID
NO. 82 HKI B9
LPNVCAFPMEKGPCQTYMTRWFFNFETGECELFAYGGCGGNSNNFLRKEKCEKFCKFT SEQ ID
NO. 124 DPI-6.1
lpnvcafpmeTgpcRARFtrwffnfetgecelfayggcggnsnnflrkekcekfckft SEQ ID
NO. 125 DPI-6.2
lpnvcafpmeTgpcRARFDrwffnfetgecelfVyggcggnsnnflrkekcekfckft SEQ ID
NO. 126 DPI-4.2.2
tvaacnlpivTgpcraRFqlwafdavkgkcvlfpyggcqgngnkfysekecreycgvp SEQ ID
NO. 130 DPI-4.2.3
tvaacnlpivTgpcraRFqRwafdavkgkcvlfpyggcqgngnkfysekecreycgvp SEQ ID
NO. 131 DPI-4.2.4
tvaacnlpivTgpcraRFqRwafdavkgkcvlfVyggcqgngnkfysekecreycgvp SEQ ID
NO. 132 DPI-4.2.5
tvaacnlpiETgpcraRFDRwafdavkgkcETfVyggccgngnkfysekecreycgvp SEQ ID
NO. 133 DPI-7.1
rpdfcleppytgpckarFiryfynakaglcqtfvyggcrakrnnfksaedcmrtcgga SEQ ID
NO. 134 DPI-7.2
rpdfcleppytgpcRarFiryfyrnkaglcqtfvyggcrakrnnfksaedcmrtcgga SEQ ID
NO. 135 DPI-7.3
rpdfcleppytgpcRarFiryfynakaglcqtfvyggcGakrnnfksaedcmrtcgga SEQ ID
NO. 136 DPI-7.4
rpdfcleppDtgpcRarFDryfynakaglcEtfvyggcGakrnnfksaedcmrtcgga SEQ ID
NO. 137 Under "Affinity", "(A)" means the K.sub.D is likely to be
less than that of BPTI (viz.300 pM), "(B)" means K.sub.D is likely
to be less than 2 nM, and "(C)" means that K.sub.D is likely to be
less than 20 nM.
TABLE-US-00006 TABLE 5 vgDNA for LACI-D1 to vary residues 10, 11,
13, 15, 16, 17, & 19 for plasmin in view of App-I (now known
not to be very potent) N|K M H S F C A F K A D|E 1 2 3 4 5 6 7 8 9
10 5'- cctcct atgcat|tcc|ttc|tgc|gcc|ttc|aag|gct|RaS|- | NsiI | C|W
F|Y L|P Q|H M|I N|T N|S T S|K I|T N|K V|R A|G A|P I|M D|A D|V G S|T
C K|R A|G R|S F|I E|G R 11 12 13 14 15 16 17 18 19 20
|RNt|ggt|Nct|tgt|aRa|gSt|aNS|wtc|NNS|cgt| F|C L|W F F N I F T R 21
22 23 24 25 26 27 28 |tKS|ttc|ttc|aac|atc|ttc|acg cgt tccctcc-3'
(SEQ ID NO. 83) 3'-g aag ttg tag aag tgc gca agggagg-5' (SEQ ID NO.
84) Tm - 80.degree. C. | MluI | DNA: 262,144 * 4 = 1,048,576
protein: 143,360 * 4 = 573,440 The amino acid seq has SEQ ID NO.
85.
[0157] This variegation allows the AppI sequence to appear in the
P6-P6' positions.
TABLE-US-00007 TABLE 6 LACI-K1 derivatives selected for Plasmin
binding 1 1 1 1 1 1 1 1 1 1 2 2 Phage K.sub.D Ident 0 1 2 3 4 5 6 7
8 9 0 1 DIFFS Binding (pM) SEQ ID NO. Con- E T G P C R A R F E R W
0 SEQ ID NO. 88 sensus LACI- d d g p c k a i m k r f 7 SEQ ID NO. 2
K1 SPI31 - - - - - - - - - G - - 1 SEQ ID NO. 89 SPI11 - - - - - -
- - - D - - 1 3.2 X 88 SEQ ID NO. 40 SPI15 - S - - - - - - - D - -
2 2.5 X SEQ ID NO. 90 SPI24 D - - - - - G - - - - L 3 SEQ ID NO. 91
SPI33 - - - S - - G - - D - - 3 SEQ ID NO. 92 SPI34 - V - - - - - S
- P - - 3 SEQ ID NO. 93 SPI26 - - - - - - - T - P - F 3 SEQ ID NO.
94 SPI37 - V - - - - - S - H - - 3 SEQ ID NO. 95 SPI32 D - - - - -
- S - G - - 3 SEQ ID NO. 96 SPI12 - - - - - - G M - P - - 3 SEQ ID
NO. 97 SPI36 - G - - - - - - - N - F 3 SEQ ID NO. 98 SPI08 D G - -
- - - - - - - F 3 2.6 X SEQ ID NO. 42 SPI38 - - - - - - - - I S - F
3 SEQ ID NO. 99 SPI18 - G - - - - - K - - - F 3 SEQ ID NO. 100
SPI23 - G - - - - - K - Q - - 3 1.25 X SEQ ID NO. 43 SPI35 D S - A
- - G - - - - - 4 SEQ ID NO. 101 SPI02 D S - - - - G - - - - F 4
0.83 X SEQ ID NO. 102 SPI25 D - - - - - - S - P - L 4 SEQ ID NO.
103 SPI17 - V - - - - - - I Q - F 4 0.09 X SEQ ID NO. 104 SPI05 - S
- - - - - K - A - F 4 0.64 X SEQ ID NO. 105 SPI13 - G - - - - - K -
A - F 4 SEQ ID NO. 106 SPI07 D - - S - - - K I - - - 4 SEQ ID NO.
107 SPI03 D S - - - K - - - D - - 4 0.48 X SEQ ID NO. 108 SPI06 D G
- - - K G - - - - - 4 SEQ ID NO. 109 SPI16 - V - A - K G - - H - -
5 0.22 X SEQ ID NO. 110 SPI04 D G - - - - - S - P - F 5 SEQ ID NO.
111 SPI01 D S - A - - - M - H - F 6 0.25 X SEQ ID NO. 112 SPI14 D S
- A - - - K - R - - 5 SEQ ID NO. 113 SPI28 D S - T - K - - - P - F
6 SEQ ID NO. 114 SPI27 - - - - - K G K I A - F 6 SEQ ID NO. 115
SPI21 D S - A - K G K - - - - 6 0.38 X SEQ ID NO. 116 SPI22 D G - -
- K G K - P - F 7 2.0 X SEQ ID NO. 44 "Diffs" is the number of
differences from the Consensus. "Phage Binding" is the binding of
phage that display the named protein relative to binding of phage
that display BPTI.
TABLE-US-00008 TABLE 7 Variation of Residues 31, 32, 34; and 39 T R
Q C 5'-cctcct|acg|cgt|cag|tgc|- | MluI | F|S F|S Y|C Y|C L|P L|P G
H|R H|R N|D W|I W|I H|R T|M T|M Y|C N|K N|K A|V V|A V|A I|T D|G D|G
S|P C E|G E|Q E|Q F F|L Y|W G G C K|R G N Q 31 32 33 34 35 36 37 38
39 40 41 42 |NNS|NNS|ttc|NNt|tRS|ggt|ggt|tgt|RRg|ggt|aac|cag|- |
BstEII | gtcgtgctctttagcacgacctg-3' (SEQ ID NO. 86)
[0158] The amino acid sequence has SEQ ID NO. 87.
[0159] The EcoRI site is erased; thus, cleavage with EcoRI can be
used to eliminate (or at least greatly reduce) parental DNA.
[0160] There are 262,144 DNA sequences and 72,000 protein
sequences.
TABLE-US-00009 TABLE 8 Selectants for plasmin binding with
variegation of second loop 1 1 1 1 1 1 1 1 1 1 2 2 3 3 3 3 3 3 3 3
3 4 # Diffs Id 0 1 2 3 4 5 6 7 8 9 0 1 1 2 3 4 5 6 7 8 9 0 C1 C K1
Con1 E T g P c R A K F D r W E A f V Y g g C G g 10 SEQ ID NO. 45
SPI47 D A - - - - - - - E - F - E - I - - - - E - 7 (5) 5 SEQ ID
NO. 57 SPI51 D A - - - - - - - E - F T - - F - - - - - - 6 (4) 9
SEQ ID NO. 54 SPI52 D A - - - - - - - E - F - - - L - - - - - - 5
(3) 8 SEQ ID NO. 52 SPI46 D V - - - - - - - E - F - - - L - - - - E
- 6 (3) 7 SEQ ID NO. 53 SPI41 D A - - - - - R - E - F D T - L - - -
- E - 9 (6) 8 SEQ ID NO. 59 SPI42 - - - - - - G - - - - - Q G - - -
- - - - - 3 (3) 12 SEQ ID NO. 48 SPI43 - - - - - - G - - - - - - S
- H - - - - K - 4 (4) 11 SEQ ID NO. 51 SPI56 - - - - - - G - - - -
- A Q - - - - - - E - 4 (3) 11 SEQ ID NO. 50 SPI59 - - - - - - - -
- - - - N T - - - - - - - - 2 (2) 11 SEQ ID NO. 47 SPI60 - - - - -
- - - - - - - - P - - - - - - E - 2 (1) 9 SEQ ID NO. 46 SPI55 - V -
- - - - - - - - - H L - T - - - - - - 4 (4) 11 SEQ ID NO. 49 SPI49
- - - - - - - - I P - L - P - I W - - - - - 6 (6) 10 SEQ ID NO. 56
SPI57 D S - - - K G R - G - L T - - D W - - - - - 10 (8) 11 SEQ ID
NO. 60 SPI53 - - - - - K G S - - - - N V - R - - - - R - 7 (7) 11
SEQ ID NO. 58 SPI54 D S - - - - - R - - - - T R - P - - - - - - 6
(4) 10 SEQ ID NO. 55 SPI11 - - - - - - - R - - - - e e f i y g g c
e g [1] (4) 7 SEQ ID NO. 40 LACI1 d d g p c k a i m k r f e e f i y
g g c e g 10 (7) 0 SEQ ID NO. 2 See notes below. In the Table, "-"
means that the protein has the consensus (Con1) type. Con1 contains
the most common type at each position; amino acids shown in Con1
were not varied. Four positions (10, 31, 34, and 39) showed
significant toleration for a second type, leading to 15 subsidiary
consensus sequences: Con2-Con16. The column "# Diffs" shows the
number of differences from CON1 under "C1", the differences with
the closest of Con1-Con16 under "C", and the differences from
LACI-K1 under "K1". SPI11 was selected from a library in which
residues 31-39 were locked at the wild-type. SPI11 <BPTI <
SPI23 = SPI51 <SPI47 <QS4 <SPI22 <SPI54 <SPI43
Highly very potent Superior potent
TABLE-US-00010 TABLE 9 Conservative and Semiconservative
substitutions Initial Conservative Semi-conservative AA type
Category substitution substitution A Small non- G, S, T N, V, P,
(C) polar or slightly polar C free SH A, M, L, V, I F, G disulfide
nothing nothing D acidic, E, N, S, T, Q K, R, H, A hydrophilic E
acidic, D, Q, S, T, N K, R, H, A hydrophilic F aromatic W, Y, H, L,
M I, V, (C) G Gly-only nothing nothing conformation "normal" A, S,
N, T D, E, H, I, K, L, M, conformation Q, R, V H amphoteric Y, F,
K, R L, M, A, (C) aromatic I aliphatic, V, L, M, A F, Y, W, G (C)
branched .beta. carbon K basic R, H Q, N, S, T, D, E, A L aliphatic
M, I, V, A F, Y, W, H, (C) M hydrophobic L, I, V, A Q, F, Y, W,
(C), (R), (K), (E) N non-polar S, T, (D), Q, K, R hydrophilic A, G,
(E) P inflexible V, I A, (C), (D), (E), F, H, (K), L, M, N, Q, (R),
S, T, W, Y Q aliphatic N, E, A, S, T, D M, L, K, R plus amide R
basic K, Q, H S, T, E, D, A, S hydrophilic A, T, G, N D, E, R, K T
hydrophilic A, S, G, N, V D, E, R, K, I V aliphatic, I, L, M, A, T
P, (C) branched .beta. carbon W aromatic F, Y, H L, M, I, V, (C) Y
aromatic F, W, H L, M, I, V, (C)
[0161] Changing from A, F, H, I, L, M, P, V, W, or Y to C is
semiconservative if the new cysteine remains as a free thiol.
[0162] Changing from M to E, R, K is semiconservative if the ionic
tip of the new side group can reach the protein surface while the
methylene groups make hydrophobic contacts.
[0163] Changing from P to one of K, R, E, or D is semiconservative
if the side group is on or near the surface of the protein.
TABLE-US-00011 TABLE 10 Plasmin-inhibiting Kunitz domain
derivatives of LACI-K1 Consensus #1 Consensus #2 Consensus #3
Consensus #4 Position Type Status Type Status Type Status Type
Status 10 D fixed D fixed E/D S-S D/E S-S 11 D fixed D fixed T/S
G-S T/A G-S 12 G fixed G fixed G fixed G fixed 13 P Abs-S P VS-S P
VS-S P Abs-S 14 C fixed C fixed C fixed C fixed 15 K fixed K fixed
R S-S R S-S 16 A Abs-S A Abs-S A VS-S A S-S 17 R Abs-S R VS-S R/K
S-S K S-S 18 F Abs-S F Abs-S F VS-S F VS-S 19 E Abs-S E Abs-S E/P/D
S-S D/E VS-S 20 R fixed R fixed R fixed R fixed 21 F fixed F fixed
W/F weak- W/F weak- Sel Sel 31 E S-S E S-S E fixed E/t G-S 32 Q G-S
Q G-S E fixed A/T Strong for no charge, weak for type 33 F fixed F
fixed F fixed F fixed 34 -- no T/S weak I fixed V/L/I Weak
consensus 35 Y fixed Y fixed Y fixed Y S-S 39 -- no G weak E fixed
G/E some- consensus Sel. Sel. Abs-S Absolute Selection VS-S Very
Strong Selection S-S Strong Selection G-S Good Selection
TABLE-US-00012 TABLE 11 High Specificity Designed Plasmin
Inhibitors Sequence
1111111111222222222233333333334444444444555555555 Ident
1234567890123456789012345678901234567890123456789012345678 SEQ ID
NO. SPI11
mhsfcafkaETgPcRARFDrWffniftrqceefiyggcegnqnrfesleeckkmctrd SEQ ID
NO. 40 SPI11-R15A
mhsfcafkaETgPcAARFDrWffniftrqceefiyggcegnqnrfesleeckkmctrd SEQ ID
NO. 117 SPI11-R15G
mhsfcafkaETgPcGARFDrWffniftrqceefiyggcegnqnrfesleeckkmctrd SEQ ID
NO. 118 SPI11-R15N-
mhsfcafkaETgPcNARFDrWffniftrqceAfiyggcegnqnrfesleeckkmctrd SEQ ID
NO. 117 E32A
TABLE-US-00013 TABLE 12 vgDNA for LACI-D1 to vary residues 10, 11,
12, 13, 14, 15, 16, 17, 19, 20, 21, 37, 38, and 39 for plasmin in
view of App-I and SPI11 ##STR00001## ##STR00002##
TABLE-US-00014 TABLE 14 Definition of a Kunitz Domain (SEQ ID NO.
123) 1 2 3 4 5
1234567890123456789012345678901234567890123456789012345678
xxxxCxxxxxxGxCxxxxxxXXXxxxxxxCxxFxXXGCxXxxXxXxxxxxCxxxCxxx Where:
X1, X2, X3, X4, X58, X57, and X56 may be absent, X21 = Phe, Tyr,
Trp, X22 = Tyr or Phe, X23 = Tyr or Phe, X35 = Tyr or Trp, X36 =
Gly or Ser, X40 = Gly or Ala, X43 = Asn or Gly, and X45 = Phe or
Tyr
TABLE-US-00015 TABLE 15 Substitutions to confer high affinity for
plasmin on KuDoms Position Allowed types Position Allowed types 10
Asp, Glu, Tyr 20 Arg 11 Thr, Ala, Ser, Val, Asp 21 Phe, Trp, Tyr 12
Gly 31 Asp, Glu, Thr, Val, Gln, Ala 13 Pro, Leu, Ala 32 Thr, Ala,
Glu, Pro, Gln 14 Cys 34 Val, Ile, Thr, Leu, Phe, Tyr, His, Asp,
Ala, Ser 15 Arg, Lys 35 Tyr, Trp 16 Ala, Gly 36 Gly 17 Arg, Lys,
Ser 37 Gly 18 Phe, Ile 38 Cys 19 Glu, Asp, Pro, Gly, Ser, Ile 39
Glu, Gly, Asp, Arg, Ala, Gln, Leu, Lys, Met
[0164] In Table 15 the bold residue types are preferred
TABLE-US-00016 TABLE 16 Summary of Sequences sdected from First
LACI-K1 library for binding to Plasmin BPTI # Residues Allowed
Preferred (BPTI type) (LACI-K1) in Library Residues 13 (P) P LHPR
PL 16 (A) A AG AG 17 (R) I FYLHINA RS SCPRTVD G 18 (I) M all F 19
(I) K LWQMKAG EQ SPRTVE 31 (Q) E EQ EQ 32 (T) E EQ QE 34 (V) I all
TYHDRAVILSF 39 (R) E all GADRQFEMLVKNH
TABLE-US-00017 TABLE 17 Distribution of sequences selected from
first library: Position A C D E F G H I K L M N P Q R S T V W Y 13
x x x x x x 0 x x 1 x x 31* x 0 x x x x x 16 31* x x x x 1 x x x x
x x x x x x x x x x 17 0 0 0 x 0 0 0 0* x 0 x 0 0 x 30 2 0 0 x 0 18
0 0 0 0 32 0 0 0 0 0 0* 0 0 0 0 0 0 0 0 0 19 0 x x 31 x 0 x x 0* 0
0 x 0 1 0 0 0 0 0 x 31 x x x 28* x x x x x x x x x 4 x x x x x x 32
x x x 9* x x x x x x x x x 23 x x x x x x 34 2 0 1 0 1 0 2 5* 0 2 0
0 0 0 1 3 8 5 0 2 39 3 0 3 2* 1 10 1 0 2 2 2 1 0 3 1 0 0 1 0 0
TABLE-US-00018 TABLE 18 Distribution or amino-acid types at varied
residues in proteins selected for plasmin binding from third
library. Position A C D E F G H I K L M N P Q R S T V W Y 10 x x 7*
8 x x x x 0 x x 0 x x x x x x x x 11 4 x 0* x x 0 x 0 x x x 0 x x x
2 7 2 x x 13 0 x x x x x x x x x x x 15* x x 0 0 x x x 15 x x x x x
x x x 2* x x x x x 13 x x x x x 16 10* x x x x 5 x x x x x x x x x
x x x x x 17 x x x x x x x 0* 11 x 0 0 x x 3 1 0 x x x 18 x x x x
14 x x 1 x x x* x x x x x x x x x 19 0 0 8 5 0 1 0 0 0* 0 0 0 1 0 0
0 0 0 0 0 21 x 0 x x 5* x x x x 2 x x x x x x x x 8 x 31 1 0 1 6* 0
0 1 0 0 0 0 2 0 1 0 0 3 0 0 0 32 4 0 0 1* 0 1 0 0 0 1 0 0 2 1 1 1 2
1 0 0 34 0 0 1 x 1 0 1 2* x 3 x 0 1 x 1 0 1 4 x 0 35 x 0 x x x x x
x x x x x x x x x x x 2 13* 39 x x x 5* x 8 x x 1 x x x x x 1 x x x
x x
TABLE-US-00019 TABLE 23 Specificity Results Obtained with KuDoms
Displayed on gIIIp of M13 Target KuDom Trypsin, 2 Displayed Plasmin
Thrombin Kallikrein Trypsin washes LACI-K1 1.0 1.0 1.0 1.0 1.0 QS4
52. 0.7 0.9 4.5 0.5 BPTI 88. 1.1 1.7 0.3 0.8 LACI-K1 phage for each
Target was taken as unit binding and the other display phage are
shown as relative binding. BPTI::III phage are not easily liberated
from trypsin.
TABLE-US-00020 TABLE 24 Mat .alpha. S. cerevisiae expression
vectors: Mat.alpha.1 (Mf.alpha.8) K R P R
5'-...|AAA|AGG|CCT|CGA|G...-3' | StuI | | XhoI | Mat.alpha.2 (after
introduction of a linker into StuI-cut DNA) K R E A A E P W G A . .
L E 5'|AAA|AGG|GAA|GCG|GCC|GAG|CCA|TGG|GGC|GCC|TAA|TAG|CTC|GAG|3' |
EagI | | StyI | KasI | | XhoI | Mat.alpha.-LACI-K1 a b c d 1 2 3 4
5 6 7 8 K R E A A E M H S F C A F K
5'|AAA|AGG|GAA|GCG|GCC|GAG|atg|cat|tcc|ttc|tgc|gct|ttc|aaa| | EagI
| | NsiI | 9 10 11 12 13 14 15 16 17 18 19 20 A D D G P C K A I M K
R |gct|gat|gaC|ggT|ccG|tgt|aaa|gct|atc|atg|aaa|cgt| |RsrII | |
BspHI| 21 22 23 24 25 26 27 28 29 30 F F F N I F T R Q C
|ttc|ttc|ttc|aac|att|ttc|acG|cgt|cag|tgc| | MluI | 31 32 33 34 35
36 37 38 39 40 41 42 E E F I Y G G C E G N Q
|gag|gaA|ttC|att|tac|ggt|ggt|tgt|gaa|ggt|aac|cag| | EcoRI | |
BstEII | 43 44 45 46 47 48 49 50 N R F E S L E E
|aac|cgG|ttc|gaa|tct|ctA|gag|gaa| | | BstBI | | XbaI | | AgeI | 51
52 53 54 55 56 57 58 59 60 C K K M C T R D G A
|tgt|aag|aag|atg|tgc|act|cgt|gac|ggc|gcc|TAA|TAG|CTC|GAG|-3' | KasI
| | XhoI |
[0165] We expect that Mat.alpha. pre sequence is cleaved before
GLU.sub.a-ALA.sub.5-
CITATIONS
[0166] ADEL86: Adelman et al. Blood (1986) 68(6)1280-1284. [0167]
ANBA88: Anba et al., Biochimie (1988) 70(6)727-733. [0168] AUER88:
Auerswald et al., Bio Chem Hoppe-Seyler (1988),
369(Supplement):27-35. [0169] BANE90: Baneyx & Georgiou, J
Bacteriol (1990) 172(1)491-494. [0170] BANE91: Baneyx &
Georgiou, J Bacteriol (1991) 173(8)2696-2703. [0171] BROW91: Browne
et al., GeneBank entry M74220. [0172] BROZ90: Broze et al.,
Biochemistry (1990) 29:7539-7546. [0173] COLM87: Colman et al.,
Editors, Hemostasis and Thrombosis, Second Edition, 1987, J. B.
Lippincott Company, Philadelphia, Pa. [0174] DENN94a: Dennis &
Lazarus, J Biological Chem (1994) 269:22129-22136. [0175] DENN94b:
Dennis & Lazarus, J Biological Chem (1994) 269:22137-22144.
[0176] EIGE90: Eigenbrot et al. Protein Engineering (1990):
3(7)591-598. [0177] ELLI92: Ellis et al., Ann N Y Acad Sci (1992)
667:13-31. [0178] FIDL94: Fidler & Elis, Cell (1994)79:185-188.
[0179] FRAE89: Fraedrich et al., Thorac Cardiovasc Surg (1989)
37(2)89-91. [0180] GARD93: Gardell, Toxicol Pathol
(1993)21(2)190-8. [0181] GIRA89: Girard et al., Nature (1989),
338:518-20. [0182] GIRA91: Girard et al., J BIOL. CHEM. (1991)
266:5036-5041. [0183] GREG93: Gregg et al., Bio/Technology (1993)
11:905-910. [0184] HOOV93: Hoover et al., Biochemistry (1993)
32:10936-43. [0185] HYNE90: Hynes et al., Biochemistry (1990),
29:10018-10022. [0186] KIDO88: Kido et al., J Biol Chem (1988),
263:18104-7. [0187] KIDO90: Kido et al., Biochem & Biophys Res
Comm (1990), 167(2)716-21. [0188] KURJ82: Kurjan and Herskowitz,
Cell (1982) 30:933-943. [0189] LASK80: Laskowski & Kato, Ann
Rev Biochem (1980), 49:593-626. [0190] LEAT91: Leatherbarrow &
Salacinski, Biochemistry (1991) 30(44)10717-21. [0191] LOHM93:
Lohmann & J Marshall, Refract Corneal Surg (1993) 9(4)300-2.
[0192] LUCA83: Lucas et al., J Biological Chem (1983)
258(7)4249-56. [0193] MANN87: Mann & Foster, Chapter 10 of
COLM87. [0194] MIYA85: Miyajima et al., Gene (1985) 37:155-161.
[0195] NEUH89: Neuhaus et al., Lancet (1989) 2(8668)924-5. [0196]
NOVO89: Novotny et al., J. BIOL. CHEM. (1989) 264:18832-18837.
[0197] OREI94: O'Reilly et al., Cell (1994) 79:315-328. [0198]
PARK86: Park & Tulinsky, Biochemistry (1986) 25(14)3977-3982.
[0199] PUTT89: Putterman, Acta Chir Scand (1989) 155(6-7)367.
[0200] ROBB87: Robbins, Chapter 21 of COLM87 [0201] SCHE67:
Schechter & Berger. Biochem Biophys Res Commun (1967)
27:157-162. [0202] SCHE68: Schechter & Berger. Biochem Biophys
Res Commun (1968) 32:898-902. [0203] SCHN86: Schnabel et al., Biol
Chem Hoppe-Syler (1986), 367: 1167-76. [0204] SHER89: Sheridan et
al., Dis Colon Rectum (1989) 32(6)505-8. [0205] SPRE94: Sprecher et
al., Proc Natl Acad Sci USA 91:3353-3357 (1994) [0206] VAND92: van
Diji et al., EMBO J (1992) 11(8)2819-2828. [0207] VARA83: Varadi
& Patthy, Biochemistry (1983) 22:2440-2446. [0208] VARA84:
Varadi & Patthy, Biochemistry (1984) 23:2108-2112. [0209]
VEDV91: Vedvick et al., J Ind Microbiol (1991) 7:197-201. [0210]
WAGN92: Wagner et al., Biochem Biophys Res Comm (1992)
186:1138-1145. [0211] WUNT88: Wun et al., J. BIOL. CHEM. (1988)
263:6001-6004.
Sequence CWU 1
1
1531304PRTHomo sapiens 1Met Ile Tyr Thr Met Lys Lys Val His Ala Leu
Trp Ala Ser Val Cys1 5 10 15Leu Leu Leu Asn Leu Ala Pro Ala Pro Leu
Asn Ala Asp Ser Glu Glu 20 25 30Asp Glu Glu His Thr Ile Ile Thr Asp
Thr Glu Leu Pro Pro Leu Lys 35 40 45Leu Met His Ser Phe Cys Ala Phe
Lys Ala Asp Asp Gly Pro Cys Lys 50 55 60Ala Ile Met Lys Arg Phe Phe
Phe Asn Ile Phe Thr Arg Gln Cys Glu65 70 75 80Glu Phe Ile Tyr Gly
Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser 85 90 95Leu Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp Asn Ala Asn Arg Ile 100 105 110Ile Lys
Thr Thr Leu Gln Gln Glu Lys Pro Asp Phe Cys Phe Leu Glu 115 120
125Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg Tyr Phe Tyr Asn
130 135 140Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys Tyr Gly Gly Cys
Leu Gly145 150 155 160Asn Met Asn Asn Phe Glu Thr Leu Glu Glu Cys
Lys Asn Ile Cys Glu 165 170 175Asp Gly Pro Asn Gly Phe Gln Val Asp
Asn Tyr Gly Thr Gln Leu Asn 180 185 190Ala Val Asn Asn Ser Leu Thr
Pro Gln Ser Thr Lys Val Pro Ser Leu 195 200 205Phe Glu Phe His Gly
Pro Ser Trp Cys Leu Thr Pro Ala Asp Arg Gly 210 215 220Leu Cys Arg
Ala Asn Glu Asn Arg Phe Tyr Tyr Asn Ser Val Ile Gly225 230 235
240Lys Cys Arg Pro Phe Lys Tyr Ser Gly Cys Gly Gly Asn Glu Asn Asn
245 250 255Phe Thr Ser Lys Gln Glu Cys Leu Arg Ala Cys Lys Lys Gly
Phe Ile 260 265 270Gln Arg Ile Ser Lys Gly Gly Leu Ile Lys Thr Lys
Arg Lys Arg Lys 275 280 285Lys Gln Arg Val Lys Ile Ala Tyr Glu Glu
Ile Phe Val Lys Asn Met 290 295 300258PRTHomo sapiens 2Met His Ser
Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Ile Met
Lys Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30Phe
Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55358PRTArtificial
SequenceSynthetically generated peptide 3Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Thr Tyr Gly
Gly Cys Arg Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 55458PRTArtificial
SequenceSynthetically generated peptide 4Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Tyr Tyr Gly
Gly Cys Asp Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 55558PRTArtificial
SequenceSynthetically generated peptide 5Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe His Tyr Gly
Gly Cys Asp Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 55658PRTArtificial
SequenceSynthetically generated peptide 6Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Asp Tyr Gly
Gly Cys Ala Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 55758PRTArtificial
SequenceSynthetically generated peptide 7Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Gln Glu 20 25 30Phe Arg Tyr Gly
Gly Cys Asp Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 55858PRTArtificial
SequenceSynthetically generated peptide 8Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Gln Gln 20 25 30Phe Tyr Tyr Gly
Gly Cys Gln Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 55958PRTArtificial
SequenceSynthetically generated peptide 9Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30Phe Ala Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 551058PRTArtificial
SequenceSynthetically generated peptide 10Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Gln Gln 20 25 30Phe Val Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 551158PRTArtificial
SequenceSynthetically generated peptide 11Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Thr Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 551258PRTArtificial
SequenceSynthetically generated peptide 12Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30Phe Thr Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 551358PRTArtificial
SequenceSynthetically generated peptide 13Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Ile Tyr Gly
Gly Cys Gln Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 551458PRTArtificial
SequenceSynthetically generated peptide 14Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Ile Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 551558PRTArtificial
SequenceSynthetically generated peptide 15Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Ile Tyr Gly
Gly Cys Phe Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 551658PRTArtificial
SequenceSynthetically generated peptide 16Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Gln Gln 20 25 30Phe His Tyr Gly
Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 551758PRTArtificial
SequenceSynthetically generated peptide 17Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Val Tyr Gly
Gly Cys Ala Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 551858PRTArtificial
SequenceSynthetically generated peptide 18Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Leu Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 551958PRTArtificial
SequenceSynthetically generated peptide 19Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Ile Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 552058PRTArtificial
SequenceSynthetically generated peptide 20Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Val Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 552158PRTArtificial
SequenceSynthetically generated peptide 21Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30Phe Val Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 552258PRTArtificial
SequenceSynthetically generated peptide 22Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Leu Cys Lys Gly1 5 10 15Arg Phe Gln Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30Phe Ile Tyr Gly
Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 552358PRTArtificial
SequenceSynthetically generated peptide 23Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Thr Tyr Gly
Gly Cys Met Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 552458PRTArtificial
SequenceSynthetically generated peptide 24Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Ser Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 552558PRTArtificial
SequenceSynthetically generated peptide 25Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30Phe Leu Tyr Gly
Gly Cys Leu Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 552658PRTArtificial
SequenceSynthetically generated peptide 26Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Ser Tyr Gly
Gly Cys Gln Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 552758PRTArtificial
SequenceSynthetically generated peptide 27Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Ala Tyr Gly
Gly Cys Ala Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 552858PRTArtificial
SequenceSynthetically generated peptide 28Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Ile Tyr Gly
Gly Cys Val Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 552958PRTArtificial
SequenceSynthetically generated peptide 29Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30Phe Ser Tyr Gly
Gly Cys Lys Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 553058PRTArtificial
SequenceSynthetically generated peptide 30Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30Phe Val Tyr Gly
Gly Cys Lys Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 553158PRTArtificial
SequenceSynthetically generated peptide 31Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Ser Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Thr Tyr Gly
Gly Cys Asn Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 553258PRTArtificial
SequenceSynthetically generated peptide 32Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Ser Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30Phe Thr Tyr Gly
Gly Cys Leu Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 553358PRTArtificial
SequenceSynthetically generated peptide 33Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Phe Tyr Gly
Gly Cys His Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 553458PRTArtificial
SequenceSynthetically generated peptide 34Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Glu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30Phe Thr Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 553558PRTArtificial
SequenceSynthetically generated
peptide 35Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys
Lys Ala1 5 10 15Arg Phe Glu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln
Cys Glu Gln 20 25 30Phe Thr Tyr Gly Gly Cys Met Gly Asn Gln Asn Arg
Phe Glu Ser Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
553658PRTArtificial SequenceSynthetically generated peptide 36Met
His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10
15Arg Phe Glu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln
20 25 30Phe Thr Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
553758PRTArtificial SequenceSynthetically generated peptide 37Met
His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10
15Arg Phe Glu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln
20 25 30Phe Val Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 553858PRTBos
taurus 38Arg Pro Asp Phe Cys Leu Glu Pro Pro Tyr Thr Gly Pro Cys
Lys Ala1 5 10 15Arg Ile Ile Arg Tyr Phe Tyr Asn Ala Lys Ala Gly Leu
Cys Gln Thr 20 25 30Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn Asn
Phe Lys Ser Ala 35 40 45Glu Asp Cys Met Arg Thr Cys Gly Gly Ala 50
553958PRTHomo sapiens 39Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr
Gly Pro Cys Arg Ala1 5 10 15Met Ile Ser Arg Trp Tyr Phe Asp Val Thr
Glu Gly Lys Cys Ala Pro 20 25 30Phe Phe Tyr Gly Gly Cys Gly Gly Asn
Arg Asn Asn Phe Asp Thr Glu 35 40 45Glu Tyr Cys Met Ala Val Cys Gly
Ser Ala 50 554058PRTArtificial SequenceSynthetically generated
peptide 40Met His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Pro Cys
Arg Ala1 5 10 15Arg Phe Asp Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln
Cys Glu Glu 20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg
Phe Glu Ser Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
554158PRTArtificial SequenceSynthetically generated peptide 41Met
His Ser Phe Cys Ala Phe Lys Ala Glu Ser Gly Pro Cys Arg Ala1 5 10
15Arg Phe Asp Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
554258PRTArtificial SequenceSynthetically generated peptide 42Met
His Ser Phe Cys Ala Phe Lys Ala Asp Gly Gly Pro Cys Arg Ala1 5 10
15Arg Phe Glu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
554358PRTArtificial SequenceSynthetically generated peptide 43Met
His Ser Phe Cys Ala Phe Lys Ala Glu Gly Gly Pro Cys Arg Ala1 5 10
15Lys Phe Gln Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
554458PRTArtificial SequenceSynthetically generated peptide 44Met
His Ser Phe Cys Ala Phe Lys Ala Asp Gly Gly Pro Cys Lys Gly1 5 10
15Lys Phe Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
554558PRTArtificial SequenceSynthetically generated peptide 45Met
His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Pro Cys Arg Ala1 5 10
15Lys Phe Asp Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Ala
20 25 30Phe Val Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
554658PRTArtificial SequenceSynthetically generated peptide 46Met
His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Pro Cys Arg Ala1 5 10
15Lys Phe Asp Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Pro
20 25 30Phe Val Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
554758PRTArtificial SequenceSynthetically generated peptide 47Met
His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Pro Cys Arg Ala1 5 10
15Lys Phe Asp Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Asn Thr
20 25 30Phe Val Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
554858PRTArtificial SequenceSynthetically generated peptide 48Met
His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Pro Cys Arg Gly1 5 10
15Lys Phe Asp Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Gln Gly
20 25 30Phe Val Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
554958PRTArtificial SequenceSynthetically generated peptide 49Met
His Ser Phe Cys Ala Phe Lys Ala Glu Val Gly Pro Cys Arg Ala1 5 10
15Lys Phe Asp Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys His Leu
20 25 30Phe Thr Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
555058PRTArtificial SequenceSynthetically generated peptide 50Met
His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Pro Cys Arg Gly1 5 10
15Lys Phe Asp Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Ala Gln
20 25 30Phe Val Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
555158PRTArtificial SequenceSynthetically generated peptide 51Met
His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Pro Cys Arg Gly1 5 10
15Lys Phe Asp Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Ser
20 25 30Phe His Tyr Gly Gly Cys Lys Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
555258PRTArtificial SequenceSynthetically generated peptide 52Met
His Ser Phe Cys Ala Phe Lys Ala Asp Ala Gly Pro Cys Arg Ala1 5 10
15Lys Phe Glu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Ala
20 25 30Phe Leu Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
555358PRTArtificial SequenceSynthetically generated peptide 53Met
His Ser Phe Cys Ala Phe Lys Ala Asp Val Gly Pro Cys Arg Ala1 5 10
15Lys Phe Glu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Ala
20 25 30Phe Leu Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
555458PRTArtificial SequenceSynthetically generated peptide 54Met
His Ser Phe Cys Ala Phe Lys Ala Asp Ala Gly Pro Cys Arg Ala1 5 10
15Lys Phe Glu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Thr Ala
20 25 30Phe Phe Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
555558PRTArtificial SequenceSynthetically generated peptide 55Met
His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Pro Cys Arg Ala1 5 10
15Arg Phe Asp Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Thr Arg
20 25 30Phe Pro Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
555658PRTArtificial SequenceSynthetically generated peptide 56Met
His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Pro Cys Arg Ala1 5 10
15Lys Ile Pro Arg Leu Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Pro
20 25 30Phe Ile Trp Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
555758PRTArtificial SequenceSynthetically generated peptide 57Met
His Ser Phe Cys Ala Phe Lys Ala Asp Ala Gly Pro Cys Arg Ala1 5 10
15Lys Phe Glu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
555858PRTArtificial SequenceSynthetically generated peptide 58Met
His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Pro Cys Lys Gly1 5 10
15Ser Phe Asp Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Asn Val
20 25 30Phe Arg Tyr Gly Gly Cys Arg Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
555958PRTArtificial SequenceSynthetically generated peptide 59Met
His Ser Phe Cys Ala Phe Lys Ala Asp Ala Gly Pro Cys Arg Ala1 5 10
15Arg Phe Glu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Asp Thr
20 25 30Phe Leu Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
556058PRTArtificial SequenceSynthetically generated peptide 60Met
His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Pro Cys Lys Gly1 5 10
15Arg Phe Gly Arg Leu Phe Phe Asn Ile Phe Thr Arg Gln Cys Thr Ala
20 25 30Phe Asp Trp Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
556158PRTArtificial SequenceSynthetically generated peptide 61Met
His Ser Phe Cys Ala Phe Lys Ala Asp Thr Gly Pro Cys Arg Ala1 5 10
15Arg Phe Asp Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Ala
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
556258PRTArtificial SequenceSynthetically generated peptide 62Met
His Ser Phe Cys Ala Phe Lys Ala Asp Thr Gly Pro Cys Arg Ala1 5 10
15Arg Phe Asp Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
556358PRTArtificial SequenceSynthetically generated peptide 63Met
His Ser Phe Cys Ala Phe Lys Ala Asp Ala Gly Pro Cys Arg Ala1 5 10
15Arg Phe Asp Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
556458PRTHomo sapiens 64Lys Pro Asp Phe Cys Phe Leu Glu Glu Asp Pro
Gly Ile Cys Arg Gly1 5 10 15Tyr Ile Thr Arg Tyr Phe Tyr Asn Asn Gln
Thr Lys Gln Cys Glu Arg 20 25 30Phe Lys Tyr Gly Gly Cys Leu Gly Asn
Met Asn Asn Phe Glu Thr Leu 35 40 45Glu Glu Cys Lys Asn Ile Cys Glu
Asp Gly 50 556558PRTArtificial SequenceSynthetically generated
peptide 65Lys Pro Asp Phe Cys Phe Leu Glu Glu Asp Thr Gly Pro Cys
Arg Gly1 5 10 15Arg Phe Asp Arg Tyr Phe Tyr Asn Asn Gln Thr Lys Gln
Cys Glu Thr 20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Met Asn Asn
Phe Glu Thr Leu 35 40 45Glu Glu Cys Lys Asn Ile Cys Glu Asp Gly 50
556658PRTHomo sapiens 66Gly Pro Ser Trp Cys Leu Thr Pro Ala Asp Arg
Gly Leu Cys Arg Ala1 5 10 15Asn Glu Asn Arg Phe Tyr Tyr Asn Ser Val
Ile Gly Lys Cys Arg Pro 20 25 30Phe Lys Tyr Ser Gly Cys Gly Gly Asn
Glu Asn Asn Phe Thr Ser Lys 35 40 45Gln Glu Cys Leu Arg Ala Cys Lys
Lys Gly 50 556758PRTArtificial SequenceSynthetically generated
peptide 67Gly Pro Ser Trp Cys Leu Thr Pro Ala Asp Thr Gly Pro Cys
Arg Ala1 5 10 15Arg Phe Asp Arg Phe Tyr Tyr Asn Ser Val Ile Gly Lys
Cys Glu Pro 20 25 30Phe Ile Tyr Gly Gly Cys Gly Gly Asn Glu Asn Asn
Phe Thr Ser Lys 35 40 45Gln Glu Cys Leu Arg Ala Cys Lys Lys Gly 50
556858PRTHomo sapiens 68Glu Thr Asp Ile Cys Lys Leu Pro Lys Asp Glu
Gly Thr Cys Arg Asp1 5 10 15Phe Ile Leu Lys Trp Tyr Tyr Asp Pro Asn
Thr Lys Ser Cys Ala Arg 20 25 30Phe Trp Tyr Gly Gly Cys Gly Gly Asn
Glu Asn Lys Phe Gly Ser Gln 35 40 45Lys Glu Cys Glu Lys Val Cys Ala
Pro Val 50 556958PRTArtificial SequenceSynthetically generated
peptide 69Glu Thr Asp Ile Cys Lys Leu Pro Lys Asp Thr Gly Pro Cys
Arg Ala1 5 10 15Arg Phe Asp Lys Trp Tyr Tyr Asp Pro Asn Thr Lys Ser
Cys Glu Pro 20 25 30Phe Val Tyr Gly Gly Cys Gly Gly Asn Glu Asn Lys
Phe Gly Ser Gln 35 40 45Lys Glu Cys Glu Lys Val Cys Ala Pro Val 50
557058PRTHomo sapiens 70Asn Ala Glu Ile Cys Leu Leu Pro Leu Asp Tyr
Gly Pro Cys Arg Ala1 5 10 15Leu Leu Leu Arg Tyr Tyr Tyr Asp Arg Tyr
Thr Gln Ser Cys Arg Gln 20 25 30Phe Leu Tyr Gly Gly Cys Glu Gly Asn
Ala Asn Asn Phe Tyr Thr Trp 35 40 45Glu Ala Cys Asp Asp Ala Cys Trp
Arg Ile 50 557158PRTArtificial SequenceSynthetically generated
peptide 71Asn Ala Glu Ile Cys Leu Leu Pro Leu Asp Thr Gly Pro Cys
Arg Ala1 5 10 15Arg Phe Asp Arg Tyr Tyr Tyr Asp Arg Tyr Thr Gln Ser
Cys Glu Gln 20 25 30Phe Leu Tyr Gly Gly Cys Glu Gly Asn Ala Asn Asn
Phe Tyr Thr Trp 35 40 45Glu Ala Cys Asp Asp Ala Cys Trp Arg Ile 50
557261PRTHomo sapiens 72Val Pro Lys Val Cys Arg Leu Gln Val Ser Val
Asp Asp Gln Cys Glu1 5 10 15Gly Ser Thr Glu Lys Tyr Phe Phe Asn Leu
Ser Ser Met Thr Cys Glu 20 25 30Lys Phe Phe Ser Gly Gly Cys His Arg
Asn Arg Ile Glu Asn Arg Phe 35 40 45Pro Asp Glu Ala Thr Cys Met Gly
Phe Cys Ala Pro Lys 50 55 607358PRTArtificial SequenceSynthetically
generated peptide 73Val Pro Lys Val Cys Arg Leu Gln Val Glu Thr Gly
Pro Cys Arg Gly1 5 10 15Lys Phe Glu Lys Tyr Phe Phe Asn Leu
Ser Ser Met Thr Cys Glu Thr 20 25 30Phe Val Tyr Gly Gly Cys Glu Gly
Asn Arg Asn Arg Phe Pro Asp Glu 35 40 45Ala Thr Cys Met Gly Phe Cys
Ala Pro Lys 50 557458PRTHomo sapiens 74Ile Pro Ser Phe Cys Tyr Ser
Pro Lys Asp Glu Gly Leu Cys Ser Ala1 5 10 15Asn Val Thr Arg Tyr Tyr
Phe Asn Pro Arg Tyr Arg Thr Cys Asp Ala 20 25 30Phe Thr Tyr Thr Gly
Cys Gly Gly Asn Asp Asn Asn Phe Val Ser Arg 35 40 45Glu Asp Cys Lys
Arg Ala Cys Ala Lys Ala 50 557558PRTArtificial
SequenceSynthetically generated peptide 75Ile Pro Ser Phe Cys Tyr
Ser Pro Lys Asp Thr Gly Pro Cys Arg Ala1 5 10 15Arg Phe Thr Arg Tyr
Tyr Phe Asn Pro Arg Tyr Arg Thr Cys Asp Ala 20 25 30Phe Thr Tyr Gly
Gly Cys Gly Gly Asn Asp Asn Asn Phe Val Ser Arg 35 40 45Glu Asp Cys
Lys Arg Ala Cys Ala Lys Ala 50 557658PRTHomo sapiens 76Lys Glu Asp
Ser Cys Gln Leu Gly Tyr Ser Ala Gly Pro Cys Met Gly1 5 10 15Met Thr
Ser Arg Tyr Phe Tyr Asn Gly Thr Ser Met Ala Cys Glu Thr 20 25 30Phe
Gln Tyr Gly Gly Cys Met Gly Asn Gly Asn Asn Phe Val Thr Glu 35 40
45Lys Glu Cys Leu Gln Thr Cys Arg Thr Val 50 557758PRTArtificial
SequenceSynthetically generated peptide 77Lys Glu Asp Ser Cys Gln
Leu Gly Tyr Glu Ala Gly Pro Cys Arg Gly1 5 10 15Lys Phe Ser Arg Tyr
Phe Tyr Asn Gly Thr Ser Met Ala Cys Glu Thr 20 25 30Phe Val Tyr Gly
Gly Cys Gly Gly Asn Gly Asn Asn Phe Val Thr Glu 35 40 45Lys Glu Cys
Leu Gln Thr Cys Arg Thr Val 50 557858PRTHomo sapiens 78Thr Val Ala
Ala Cys Asn Leu Pro Ile Val Arg Gly Pro Cys Arg Ala1 5 10 15Phe Ile
Gln Leu Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu 20 25 30Phe
Pro Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Tyr Ser Glu 35 40
45Lys Glu Cys Arg Glu Tyr Cys Gly Val Pro 50 557958PRTArtificial
SequenceSynthetically generated peptide 79Thr Val Ala Ala Cys Asn
Leu Pro Ile Asp Thr Gly Pro Cys Arg Ala1 5 10 15Arg Phe Gln Leu Trp
Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu 20 25 30Phe Val Tyr Gly
Gly Cys Gln Gly Asn Gly Asn Lys Phe Tyr Ser Glu 35 40 45Lys Glu Cys
Arg Glu Tyr Cys Gly Val Pro 50 558058PRTHomo sapiens 80Val Arg Glu
Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala1 5 10 15Met Ile
Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro 20 25 30Phe
Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu 35 40
45Glu Tyr Cys Met Ala Val Cys Gly Ser Ala 50 558158PRTArtificial
SequenceSynthetically generated peptide 81Val Arg Glu Val Cys Ser
Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala1 5 10 15Arg Phe Ser Arg Trp
Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro 20 25 30Phe Phe Tyr Gly
Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu 35 40 45Glu Tyr Cys
Met Ala Val Cys Gly Ser Ala 50 558258PRTArtificial
SequenceSynthetically generated peptide 82Val Arg Glu Val Cys Ser
Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala1 5 10 15Arg Phe Ser Arg Trp
Tyr Phe Asp Val Thr Glu Gly Lys Cys Glu Pro 20 25 30Phe Ile Tyr Gly
Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu 35 40 45Glu Tyr Cys
Met Ala Val Cys Gly Ser Ala 50 558397DNAArtificial
SequenceSynthetically generated oligonucleotide 83cctcct atg cat
tcc ttc tgc gcc ttc aag gct ras rnt ggt nct tgt 48 Met His Ser Phe
Cys Ala Phe Lys Ala Xaa Xaa Gly Xaa Cys 1 5 10ara gst ans wtc nns
cgt tks ttc ttc aac atc ttc acg cgt 90Xaa Xaa Xaa Xaa Xaa Arg Xaa
Phe Phe Asn Ile Phe Thr Arg15 20 25tccctcc 978426DNAArtificial
SequenceSynthetically generated DNA fragment 84ggagggaacg
cgtgaagatg ttgaag 268528PRTArtificial SequenceSynthetically
generated peptide 85Met His Ser Phe Cys Ala Phe Lys Ala Xaa Xaa Gly
Xaa Cys Xaa Xaa1 5 10 15Xaa Xaa Xaa Arg Xaa Phe Phe Asn Ile Phe Thr
Arg 20 258677DNAArtificial SequenceSynthetically generated
oligonucleotide 86cctcct acg cgt cag tgc nns nns ttc nnt trs ggt
ggt tgt rrg ggt 48 Thr Arg Gln Cys Xaa Xaa Phe Xaa Xaa Gly Gly Cys
Xaa Gly 1 5 10aac cag gtcgtgctct ttagcacgac ctg 77Asn
Gln158716PRTArtificial SequenceSynthetically generated peptide
87Thr Arg Gln Cys Xaa Xaa Phe Xaa Xaa Gly Gly Cys Xaa Gly Asn Gln1
5 10 158858PRTArtificial SequenceSynthetically generated peptide
88Met His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Pro Cys Arg Ala1
5 10 15Arg Phe Glu Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu
Glu 20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu
Ser Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
558958PRTArtificial SequenceSynthetically generated peptide 89Met
His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Pro Cys Arg Ala1 5 10
15Arg Phe Gly Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
559058PRTArtificial SequenceSynthetically generated peptide 90Met
His Ser Phe Cys Ala Phe Lys Ala Glu Ser Gly Pro Cys Arg Ala1 5 10
15Arg Phe Asp Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
559158PRTArtificial SequenceSynthetically generated peptide 91Met
His Ser Phe Cys Ala Phe Lys Ala Asp Thr Gly Pro Cys Arg Gly1 5 10
15Arg Phe Glu Arg Leu Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
559258PRTArtificial SequenceSynthetically generated peptide 92Met
His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Ser Cys Arg Gly1 5 10
15Arg Phe Asp Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
559358PRTArtificial SequenceSynthetically generated peptide 93Met
His Ser Phe Cys Ala Phe Lys Ala Glu Val Gly Pro Cys Arg Ala1 5 10
15Ser Phe Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
559458PRTArtificial SequenceSynthetically generated peptide 94Met
His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Pro Cys Arg Ala1 5 10
15Thr Phe Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
559558PRTArtificial SequenceSynthetically generated peptide 95Met
His Ser Phe Cys Ala Phe Lys Ala Glu Val Gly Pro Cys Arg Ala1 5 10
15Ser Phe His Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
559658PRTArtificial SequenceSynthetically generated peptide 96Met
His Ser Phe Cys Ala Phe Lys Ala Asp Thr Gly Pro Cys Arg Ala1 5 10
15Ser Phe Gly Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
559758PRTArtificial SequenceSynthetically generated peptide 97Met
His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Pro Cys Arg Gly1 5 10
15Met Phe Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
559858PRTArtificial SequenceSynthetically generated peptide 98Met
His Ser Phe Cys Ala Phe Lys Ala Glu Gly Gly Pro Cys Arg Ala1 5 10
15Arg Phe Asn Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
559958PRTArtificial SequenceSynthetically generated peptide 99Met
His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Pro Cys Arg Ala1 5 10
15Arg Ile Ser Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5510058PRTArtificial SequenceSynthetically generated peptide 100Met
His Ser Phe Cys Ala Phe Lys Ala Glu Gly Gly Pro Cys Arg Ala1 5 10
15Lys Phe Glu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5510158PRTArtificial SequenceSynthetically generated peptide 101Met
His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Ala Cys Arg Gly1 5 10
15Arg Phe Glu Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5510258PRTArtificial SequenceSynthetically generated peptide 102Met
His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Pro Cys Arg Gly1 5 10
15Arg Phe Glu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5510358PRTArtificial SequenceSynthetically generated peptide 103Met
His Ser Phe Cys Ala Phe Lys Ala Asp Thr Gly Pro Cys Arg Ala1 5 10
15Ser Phe Pro Arg Leu Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5510458PRTArtificial SequenceSynthetically generated peptide 104Met
His Ser Phe Cys Ala Phe Lys Ala Glu Val Gly Pro Cys Arg Ala1 5 10
15Arg Ile Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5510558PRTArtificial SequenceSynthetically generated peptide 105Met
His Ser Phe Cys Ala Phe Lys Ala Glu Ser Gly Pro Cys Arg Ala1 5 10
15Lys Phe Ala Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5510658PRTArtificial SequenceSynthetically generated peptide 106Met
His Ser Phe Cys Ala Phe Lys Ala Glu Gly Gly Pro Cys Arg Ala1 5 10
15Lys Phe Ala Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5510758PRTArtificial SequenceSynthetically generated peptide 107Met
His Ser Phe Cys Ala Phe Lys Ala Asp Thr Gly Ser Cys Arg Ala1 5 10
15Lys Ile Glu Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5510858PRTArtificial SequenceSynthetically generated peptide 108Met
His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Pro Cys Lys Ala1 5 10
15Arg Phe Asp Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5510958PRTArtificial SequenceSynthetically generated peptide 109Met
His Ser Phe Cys Ala Phe Lys Ala Asp Gly Gly Pro Cys Lys Gly1 5 10
15Arg Phe Glu Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5511058PRTArtificial SequenceSynthetically generated peptide 110Met
His Ser Phe Cys Ala Phe Lys Ala Glu Val Gly Ala Cys Lys Gly1 5 10
15Arg Phe His Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5511158PRTArtificial SequenceSynthetically generated peptide 111Met
His Ser Phe Cys Ala Phe Lys Ala Asp Gly Gly Pro Cys Arg Ala1 5 10
15Ser Phe Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5511258PRTArtificial SequenceSynthetically generated peptide 112Met
His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Ala Cys Arg Ala1 5 10
15Met Phe His Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5511358PRTArtificial SequenceSynthetically generated peptide 113Met
His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Ala Cys Arg Ala1 5 10
15Lys Phe Arg Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln
Cys Glu Glu 20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg
Phe Glu Ser Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5511458PRTArtificial SequenceSynthetically generated peptide 114Met
His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Thr Cys Lys Ala1 5 10
15Arg Phe Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5511558PRTArtificial SequenceSynthetically generated peptide 115Met
His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Pro Cys Lys Gly1 5 10
15Lys Ile Ala Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5511658PRTArtificial SequenceSynthetically generated peptide 116Met
His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Ala Cys Lys Gly1 5 10
15Lys Phe Glu Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5511758PRTArtificial SequenceSynthetically generated peptide 117Met
His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Pro Cys Ala Ala1 5 10
15Arg Phe Asp Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5511858PRTArtificial SequenceSynthetically generated peptide 118Met
His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Pro Cys Gly Ala1 5 10
15Arg Phe Asp Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5511958PRTArtificial SequenceSynthetically generated peptide 119Met
His Ser Phe Cys Ala Phe Lys Ala Glu Thr Gly Pro Cys Asn Ala1 5 10
15Arg Phe Asp Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Ala
20 25 30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5512096DNAArtificial SequenceSynthetically generated DNA fragment
120cctcct atg cat tcc ttc tgc gcc ttc aag gct gas nct nnt nnt nnt
48 Met His Ser Phe Cys Ala Phe Lys Ala Xaa Xaa Xaa Xaa Xaa 1 5
10ara rnt ara ttc gns crt tks ttc ttc aac atc ttc acg cgt cag tgc
96Xaa Xaa Xaa Phe Xaa Xaa Xaa Phe Phe Asn Ile Phe Thr Arg Gln Cys15
20 25 3012171DNAArtificial SequenceSynthetically generated DNA
fragment 121cctcctccct ggttaccsny annannaccg taaacgaaag cctcgcactg
acgcgtgaag 60atgttgaaga a 7112230PRTArtificial
SequenceSynthetically generated peptide 122Met His Ser Phe Cys Ala
Phe Lys Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Phe Xaa Xaa Xaa
Phe Phe Asn Ile Phe Thr Arg Gln Cys 20 25 3012358PRTArtificial
SequenceSynthetically generated peptide 123Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Gly Xaa Cys Xaa Xaa1 5 10 15Xaa Xaa Xaa Arg Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 20 25 30Phe Xaa Xaa Xaa
Gly Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Cys
Xaa Xaa Xaa Cys Xaa Xaa Xaa 50 5512458PRTArtificial
SequenceSynthetically generated peptide 124Leu Pro Asn Val Cys Ala
Phe Pro Met Glu Lys Gly Pro Cys Gln Thr1 5 10 15Tyr Met Thr Arg Trp
Phe Phe Asn Phe Glu Thr Gly Glu Cys Glu Leu 20 25 30Phe Ala Tyr Gly
Gly Cys Gly Gly Asn Ser Asn Asn Phe Leu Arg Lys 35 40 45Glu Lys Cys
Glu Lys Phe Cys Lys Phe Thr 50 5512558PRTArtificial
SequenceSynthetically generated peptide 125Leu Pro Asn Val Cys Ala
Phe Pro Met Glu Thr Gly Pro Cys Arg Ala1 5 10 15Arg Phe Thr Arg Trp
Phe Phe Asn Phe Glu Thr Gly Glu Cys Glu Leu 20 25 30Phe Ala Tyr Gly
Gly Cys Gly Gly Asn Ser Asn Asn Phe Leu Arg Lys 35 40 45Glu Lys Cys
Glu Lys Phe Cys Lys Phe Thr 50 5512658PRTArtificial
SequenceSynthetically generated peptide 126Leu Pro Asn Val Cys Ala
Phe Pro Met Glu Thr Gly Pro Cys Arg Ala1 5 10 15Arg Phe Asp Arg Trp
Phe Phe Asn Phe Glu Thr Gly Glu Cys Glu Leu 20 25 30Phe Val Tyr Gly
Gly Cys Gly Gly Asn Ser Asn Asn Phe Leu Arg Lys 35 40 45Glu Lys Cys
Glu Lys Phe Cys Lys Phe Thr 50 5512758PRTArtificial
SequenceSynthetically generated peptide 127Met His Ser Phe Cys Ala
Phe Lys Ala Asp Thr Gly Pro Cys Lys Ala1 5 10 15Arg Phe Asp Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Ala 20 25 30Phe Ile Tyr Gly
Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 5512858PRTArtificial
SequenceSynthetically generated peptide 128Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Asp Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30Phe Ile Tyr Gly
Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 5512958PRTArtificial
SequenceSynthetically generated peptide 129Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Arg Phe Lys Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30Phe Ile Tyr Gly
Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 5513058PRTArtificial
SequenceSynthetically generated peptide 130Thr Val Ala Ala Cys Asn
Leu Pro Ile Val Thr Gly Pro Cys Arg Ala1 5 10 15Arg Phe Gln Leu Trp
Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu 20 25 30Phe Pro Tyr Gly
Gly Cys Gln Gly Asn Gly Asn Lys Phe Tyr Ser Glu 35 40 45Lys Glu Cys
Arg Glu Tyr Cys Gly Val Pro 50 5513158PRTArtificial
SequenceSynthetically generated peptide 131Thr Val Ala Ala Cys Asn
Leu Pro Ile Val Thr Gly Pro Cys Arg Ala1 5 10 15Arg Phe Gln Arg Trp
Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu 20 25 30Phe Pro Tyr Gly
Gly Cys Gln Gly Asn Gly Asn Lys Phe Tyr Ser Glu 35 40 45Lys Glu Cys
Arg Glu Tyr Cys Gly Val Pro 50 5513258PRTArtificial
SequenceSynthetically generated peptide 132Thr Val Ala Ala Cys Asn
Leu Pro Ile Val Thr Gly Pro Cys Arg Ala1 5 10 15Arg Phe Gln Arg Trp
Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu 20 25 30Phe Val Tyr Gly
Gly Cys Gln Gly Asn Gly Asn Lys Phe Tyr Ser Glu 35 40 45Lys Glu Cys
Arg Glu Tyr Cys Gly Val Pro 50 5513358PRTArtificial
SequenceSynthetically generated peptide 133Thr Val Ala Ala Cys Asn
Leu Pro Ile Glu Thr Gly Pro Cys Arg Ala1 5 10 15Arg Phe Asp Arg Trp
Ala Phe Asp Ala Val Lys Gly Lys Cys Glu Thr 20 25 30Phe Val Tyr Gly
Gly Cys Gly Gly Asn Gly Asn Lys Phe Tyr Ser Glu 35 40 45Lys Glu Cys
Arg Glu Tyr Cys Gly Val Pro 50 5513458PRTArtificial
SequenceSynthetically generated peptide 134Arg Pro Asp Phe Cys Leu
Glu Pro Pro Tyr Thr Gly Pro Cys Lys Ala1 5 10 15Arg Phe Ile Arg Tyr
Phe Tyr Asn Ala Lys Ala Gly Leu Cys Gln Thr 20 25 30Phe Val Tyr Gly
Gly Cys Arg Ala Lys Arg Asn Asn Phe Lys Ser Ala 35 40 45Glu Asp Cys
Met Arg Thr Cys Gly Gly Ala 50 5513558PRTArtificial
SequenceSynthetically generated peptide 135Arg Pro Asp Phe Cys Leu
Glu Pro Pro Tyr Thr Gly Pro Cys Arg Ala1 5 10 15Arg Phe Ile Arg Tyr
Phe Tyr Asn Ala Lys Ala Gly Leu Cys Gln Thr 20 25 30Phe Val Tyr Gly
Gly Cys Arg Ala Lys Arg Asn Asn Phe Lys Ser Ala 35 40 45Glu Asp Cys
Met Arg Thr Cys Gly Gly Ala 50 5513658PRTArtificial
SequenceSynthetically generated peptide 136Arg Pro Asp Phe Cys Leu
Glu Pro Pro Tyr Thr Gly Pro Cys Arg Ala1 5 10 15Arg Phe Ile Arg Tyr
Phe Tyr Asn Ala Lys Ala Gly Leu Cys Gln Thr 20 25 30Phe Val Tyr Gly
Gly Cys Gly Ala Lys Arg Asn Asn Phe Lys Ser Ala 35 40 45Glu Asp Cys
Met Arg Thr Cys Gly Gly Ala 50 5513758PRTArtificial
SequenceSynthetically generated peptide 137Arg Pro Asp Phe Cys Leu
Glu Pro Pro Asp Thr Gly Pro Cys Arg Ala1 5 10 15Arg Phe Asp Arg Tyr
Phe Tyr Asn Ala Lys Ala Gly Leu Cys Glu Thr 20 25 30Phe Val Tyr Gly
Gly Cys Gly Ala Lys Arg Asn Asn Phe Lys Ser Ala 35 40 45Glu Asp Cys
Met Arg Thr Cys Gly Gly Ala 50 5513813DNAArtificial
SequenceSynthetically generated DNA fragment 138aaa agg cct cga g
13Lys Arg Pro Arg11394PRTArtificial SequenceSynthetically generated
peptide 139Lys Arg Pro Arg114042DNAArtificial SequenceSynthetically
generated DNA fragment 140aaa agg gaa gcg gcc gag cca tgg ggc gcc
taa tag ctc gag 42Lys Arg Glu Ala Ala Glu Pro Trp Gly Ala * * Leu
Glu1 5 1014112PRTArtificial SequenceSynthetically generated peptide
141Lys Arg Glu Ala Ala Glu Pro Trp Gly Ala Leu Glu1 5
10142210DNAArtificial SequenceSynthetically generated
oligonucleotide 142aaa agg gaa gcg gcc gag atg cat tcc ttc tgc gct
ttc aaa gct gat 48Lys Arg Glu Ala Ala Glu Met His Ser Phe Cys Ala
Phe Lys Ala Asp1 5 10 15gac ggt ccg tgt aaa gct atc atg aaa cgt ttc
ttc ttc aac att ttc 96Asp Gly Pro Cys Lys Ala Ile Met Lys Arg Phe
Phe Phe Asn Ile Phe 20 25 30acg cgt cag tgc gag gaa ttc att tac ggt
ggt tgt gaa ggt aac cag 144Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly
Gly Cys Glu Gly Asn Gln 35 40 45aac cgg ttc gaa tct cta gag gaa tgt
aag aag atg tgc act cgt gac 192Asn Arg Phe Glu Ser Leu Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp 50 55 60ggc gcc taatagctcg ag 210Gly
Ala6514366PRTArtificial SequenceSynthetically generated peptide
143Lys Arg Glu Ala Ala Glu Met His Ser Phe Cys Ala Phe Lys Ala Asp1
5 10 15Asp Gly Pro Cys Lys Ala Ile Met Lys Arg Phe Phe Phe Asn Ile
Phe 20 25 30Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly
Asn Gln 35 40 45Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys
Thr Arg Asp 50 55 60Gly Ala65144132DNAArtificial
SequenceSynthetically generated DNA fragment 144cctcct atg cat tcc
ttc tgc gcc ttc aag gct gas nct nnt nnt nnt 48 Met His Ser Phe Cys
Ala Phe Lys Ala Xaa Xaa Xaa Xaa Xaa 1 5 10ara rnt ara ttc gns crt
tks ttc ttc aac atc ttc acg cgt cag tgc 96Xaa Xaa Xaa Phe Xaa Xaa
Xaa Phe Phe Asn Ile Phe Thr Arg Gln Cys15 20 25 30gag gct ttc gtt
tac ggt nnt nnt rns ggt aac cag 132Glu Ala Phe Val Tyr Gly Xaa Xaa
Xaa Gly Asn Gln 35 4014542PRTArtificial SequenceSynthetically
generated peptide 145Met His Ser Phe Cys Ala Phe Lys Ala Xaa Xaa
Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Phe Xaa Xaa Xaa Phe Phe Asn Ile Phe
Thr Arg Gln Cys Glu Ala 20 25 30Phe Val Tyr Gly Xaa Xaa Xaa Gly Asn
Gln 35 4014612PRTArtificial SequenceSynthetically generated peptide
146Glu Thr Gly Pro Cys Arg Ala Lys Phe Asp Arg Trp1 5
1014710PRTArtificial SequenceSyntheticaly generated peptide 147Glu
Ala Phe Val Tyr Gly Gly Cys Gly Gly1 5 1014812PRTArtificial
SequenceSynthetically generated peptide 148Asp Thr Gly Pro Cys Arg
Ala Arg Phe Asp Arg Phe1 5 1014910PRTArtificial
SequenceSynthetically generated peptide 149Glu Ala Phe Ile Tyr Gly
Gly Cys Glu Gly1 5 1015058PRTArtificial SequenceSynthetically
generated peptide 150Met His Ser Phe Cys Ala Phe Lys Ala Xaa Xaa
Gly Xaa Cys Xaa Xaa1 5 10 15Xaa Xaa Xaa Arg Trp Xaa Xaa Asn Ile Phe
Thr Arg Gln Cys Xaa Xaa 20 25 30Phe Xaa Xaa Gly Gly Cys Xaa Xaa Asn
Gln Xaa Arg Xaa Glu Ser Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr
Arg Asp 50 5515158PRTArtificial SequenceSynthetically generated
peptide 151Met His Ser Phe Cys Ala Phe Lys Ala Xaa Xaa Gly Xaa Cys
Xaa Xaa1 5 10 15Xaa Xaa Xaa Arg Xaa Xaa Xaa Asn Ile Phe Thr Arg Gln
Cys Xaa Xaa 20 25 30Phe Xaa Xaa Gly Gly Cys Xaa Xaa Asn Gln Xaa Arg
Xaa Glu Ser Leu 35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
5515256PRTArtificial SequenceSynthetically generated peptide 152Met
His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10
15Arg Phe Glu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Xaa Xaa
20 25 30Phe Xaa Tyr Gly Gly Cys Xaa Gly Asn Gln Asn Arg Phe Glu Ser
Leu 35 40 45Glu Glu Lys Met Cys Thr Arg Asp 50 5515358PRTArtificial
SequenceSynthetically generated peptide 153Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Gly Xaa Cys Xaa Xaa1 5 10 15Xaa Xaa Xaa Arg Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 20 25 30Phe Xaa Xaa Xaa
Gly Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Cys
Xaa Xaa Xaa Cys Xaa Xaa Xaa 50 55
* * * * *