U.S. patent application number 09/750964 was filed with the patent office on 2002-08-01 for kunitz domain polypeptide zkun10.
Invention is credited to Fox, Brian A., Sheppard, Paul O..
Application Number | 20020102703 09/750964 |
Document ID | / |
Family ID | 26869132 |
Filed Date | 2002-08-01 |
United States Patent
Application |
20020102703 |
Kind Code |
A1 |
Sheppard, Paul O. ; et
al. |
August 1, 2002 |
Kunitz domain polypeptide zkun10
Abstract
Proteinase inhibitors comprising a Kunitz domain are disclosed.
The Kunitz domain comprises a motif of amino acid residues as shown
in SEQ ID NO:4, and the sequence of the Kunitz domain is shown in
residues 57 through 107 of SEQ ID NO:2. The polypeptide also
includes an N-terminal collagen domain in which a von Willebrand
domain resides, and is shown in SEQ ID NO: 5. Also disclosed are
methods for making the proteinase inhibitors, and expression
vectors and cultured cells that are useful within the methods. The
proteinase inhibitors may be used as components of cell culture
media, in protein purification, and as inhibitors of protease
degradation of plasma proteins.
Inventors: |
Sheppard, Paul O.; (Granite
Falls, WA) ; Fox, Brian A.; (Seattle, WA) |
Correspondence
Address: |
Deborah A. Sawislak
ZymoGenetics, Inc
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Family ID: |
26869132 |
Appl. No.: |
09/750964 |
Filed: |
December 28, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60173425 |
Dec 29, 1999 |
|
|
|
Current U.S.
Class: |
435/226 ;
435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C07K 14/8114 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
435/226 ;
435/69.1; 435/325; 536/23.2 |
International
Class: |
C12N 009/64; C07H
021/04; C12P 021/02; C12N 005/06 |
Claims
We claim:
1. An isolated polypeptide comprising a sequence of amino acid
residues as shown in SEQ ID NO: 2 from residue 57 (Cys) to residue
107 (Cys).
2. An isolated polypeptide comprising a sequence of amino acid
residues with at least 95% identity to SEQ ID NO: 2 from residue 1
(Ile) to residue 111 (Gln).
3. The polypeptide of claim 2, wherein any variation in the
polypeptide in the region of amino acid residues 57 to 107 is
subject to the limitations as shown in SEQ ID NO: 4.
4. An isolated polypeptide comprising a sequence of amino acid
residues with at least 95% identity to SEQ ID NO: 6 from residue 1
(Met) to residue 415 (Gln).
5. The polypeptide of claim 4, wherein any variation in the
polypeptide in the region of amino acid residues 361 (Cys) to 411
(Cys) is subject to the limitations as shown in SEQ ID NO: 4.
6. An isolated polypeptide comprising a sequence of amino acid
residues as shown in SEQ ID NO: 6 from amino acid residue 1 (Met)
to amino acid residue 415 (Gln).
7. A fusion protein comprising at least two polypeptides, wherein a
first polypeptide and second polypeptide, and wherein at least one
of the polypeptides comprise a sequence of amino acid residues as
shown in SEQ ID NO: 2 from amino acid residue 57 (Cys) to amino
acid residue 107 (Cys).
8. A fusion protein comprising at least three polypeptides, wherein
a first polypeptide comprises a secretory signal polypeptide
followed by a second polypeptide comprising a collagen domain
polypeptide containing one or more von Willebrand domains, followed
by third polypeptide comprising one or more Kunitz domains, and
wherein at least one of the Kunitz domains comprise a sequence of
amino acid residues as shown in SEQ ID NO: 2 from amino acid
residue 57 (Cys) to amino acid residue 107 (Cys).
9. The fusion protein of claim 8, wherein the collagen domain
further comprises at least one globular domains.
10. An expression vector comprising the following operably linked
elements: (a) a transcription promoter; (b) a DNA segment encoding
a polypeptide according to claim 1; and (c) a transcription
terminator.
11. The expression vector of claim 10 further comprising a
secretory signal sequence operably linked to the DNA segment.
12. A cultured cell comprising the expression vector of claim
10.
13. A method of making a polypeptide comprising: culturing a cell
according to claim 12 under conditions wherein the DNA segment is
expressed; and recovering the protein encoded by the DNA
segment.
14. An antibody that specifically binds to the protein of claim
1.
15. An isolated polynucleotide molecule comprising a nucleotide
sequence encoding a polypeptide comprising a sequence of amino acid
residues as shown in SEQ ID NO: 2 from residue 57 (Cys) to residue
107 (Cys).
16. An isolated polynucleotide molecule comprising a nucleotide
sequence encoding a polypeptide comprising a sequence of amino acid
residues with at least 95% identity to SEQ ID NO: 2 from residue 1
(Ile) to residue 111 (Gln).
17. The polynucleotide molecule of claim 16, wherein any variation
in the nucleotide sequence encoding a polypeptide that falls within
the region of amino acid residues 57 to 107 is subject to the
limitations as shown in SEQ ID NO: 4 for that corresponding
region.
18. An isolated polynucleotide molecule encoding a polypeptide
comprising a sequence of amino acid residues with at least 95%
identity to SEQ ID NO: 6 from residue 1 (Met) to residue 415
(Gln).
19. The polynucleotide molecule of claim 18, wherein any variation
in the nucleotide sequence encoding a polypeptide that falls within
the region of amino acid residues 361 (Cys) to 411 (Cys) is subject
to the limitations as shown in SEQ ID NO: 4 for that corresponding
region.
20. An isolated polynucleotide molecule comprising a sequence of
polynucleotides selected from the group consisting of: (a) a
nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 169 to
nucleotide 321; (b) a nucleotide sequence as shown SEQ ID NO: 1
from nucleotide 1 to nucleotide 333; (c) a nucleotide sequence that
encodes for a polypeptide as shown in SEQ ID NO: 2 from amino acid
residue 57 to amino acid residue 107; (d) a nucleotide sequence
that encodes for a polypeptide as shown SEQ ID NO: 2 from amino
acid residue 1 to amino acid residue 111; (e) a nucleotide sequence
as shown SEQ ID NO: 5 from nucleotide 1 to nucleotide 1248; and (f)
a nucleotide sequence that encodes for a polypeptide as shown in
SEQ ID NO: 6 from amino acid residue 1 to amino acid residue
415.
21. A method of inhibiting protease degradation or activity in a
composition containing plasma proteins comprising adding a zkun10
polypeptide composition comprising a sequence of amino acid
residues as shown in SEQ ID NO: 2 from amino acid residue 57 to
amino acid residue 107 to the composition containing plasma
proteins in an amount sufficient to reduce degradation of the
composition by proteases or protease activity in the
composition.
22. The method of claim 21, wherein reduction of degradation or
activity is determined by chromogenic substrate assays or clotting
time assays.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to U.S. Provisional Application
60/173,425, filed on Dec. 29, 1999, for which claims of benefit are
made under 35 U.S.C. .sctn.120 and 35 U.S.C. .sctn.119(e)(1), and
is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] In animals, proteinases are important in wound healing,
extracellular matrix destruction, tissue reorganization, and in
cascades leading to blood coagulation, fibrinolysis, and complement
activation. Proteinases are released by inflammatory cells for
destruction of pathogens or foreign materials, and by normal and
cancerous cells as they move through their surroundings.
[0003] The activity of proteinases is regulated by inhibitors; 10%
of the proteins in blood serum are proteinase inhibitors (Roberts
et al., Critical Reviews in Eukaryotic Gene Expression 5:385-436,
1995). One family of proteinase inhibitors, the Kunitz inhibitors,
includes inhibitors of trypsin, chymotrypsin, elastase, kallikrein,
plasmin, coagulation factors XIa and IXa, and cathepsin G. These
inhibitors thus regulate a variety of physiological processes,
including blood coagulation, fibrinolysis, and inflammation.
[0004] Proteinase inhibitors regulate the proteolytic activity of
target proteinases by occupying the active site and thereby
preventing occupation by normal substrates. Although proteinase
inhibitors fall into several unrelated structural classes, they all
possess an exposed loop (variously termed an "inhibitor loop", a
"reactive core", a "reactive site", or a "binding loop") which is
stabilized by intermolecular interactions between residues flanking
the binding loop and the protein core (Bode and Huber, Eur. J.
Biochem. 204:433-451, 1992). Interaction between inhibitor and
enzyme produces a stable complex which disassociates very slowly,
releasing either virgin (uncleaved) inhibitor, or a modified
inhibitor that is cleaved at the scissile bond of the binding
loop.
[0005] One class of proteinase inhibitors, the Kunitz inhibitors,
are generally basic, low molecular weight proteins comprising one
or more inhibitory domains ("Kunitz domains"). The Kunitz domain is
a folding domain of approximately 50-60 residues which forms a
central anti-parallel beta sheet and a short C-terminal helix. This
characteristic domain comprises six cysteine residues that form
three disulfide bonds, resulting in a double-loop structure.
Between the N-terminal region and the first beta strand resides the
active inhibitory binding loop. This binding loop is disulfide
bonded through the P2 Cys residue to the hairpin loop formed
between the last two beta strands. Isolated Kunitz domains from a
variety of proteinase inhibitors have been shown to have inhibitory
activity (e.g., Petersen et al., Eur. J. Biochem. 125:310-316,
1996; Wagner et al., Biochem. Biophys. Res. Comm. 186:1138-1145,
1992; Dennis et al., J. Biol Chem. 270:25411-25417, 1995).
[0006] Proteinase inhibitors comprising one or more Kunitz domains
include tissue factor pathway inhibitor (TFPI), tissue factor
pathway inhibitor 2 (TFPI-2), amyloid .beta.-protein precursor
(A.beta.PP), aprotinin, and placental bikunin. TFPI, an extrinsic
pathway inhibitor and a natural anticoagulant, contains three
tandemly linked Kunitz inhibitor domains. The amino-terminal Kunitz
domain inhibits factor VIIa, plasmin, and cathepsin G; the second
domain inhibits factor Xa, trypsin, and chymotrypsin; and the third
domain has no known activity (Petersen et al., ibid.). TFPI-2 has
been shown to be an inhibitor of the amidolytic and proteolytic
activities of human factor VIIa-tissue factor complex, factor XIa,
plasma kallikrein, and plasmin (Sprecher et al., Proc. Natl. Acad.
Sci. USA 91:3353-3357, 1994; Petersen et al., Biochem. 35:266-272,
1996). The ability of TFPI-2 to inhibit the factor VIIa-tissue
factor complex and its relatively high levels of transcription in
umbilical vein endothelial cells, placenta and liver suggests a
specialized role for this protein in hemostasis (Sprecher et al.,
ibid.). Aprotinin (bovine pancreatic trypsin inhibitor) is a broad
spectrum Kunitz-type serine proteinase inhibitor that has been
shown to prevent activation of the clotting cascade. Aprotinin is a
moderate inhibitor of plasma kallikrein and plasmin, and blockage
of fibrinolysis and extracorporeal coagulation have been detected
in patients given aprotinin during open heart surgery (Davis and
Whittington, Drugs 49:954-983, 1995; Dietrich et al., Thorac.
Cardiovasc. Surg. 37:92-98, 1989). Aprotinin has also been used in
the treatment of septic shock, adult respiratory distress syndrome,
acute pancreatitis, hemorrhagic shock, and other conditions
(Westaby, Ann. Thorac. Surg. 55:1033-1041, 1993; Wachtfogel et al.,
J. Thorac. Cardiovasc. Surg. 106:1-10, 1993). The clinical utility
of aprotinin is believed to arise from its inhibitory activity
towards plasma kallikrein or plasmin (Dennis et al., ibid.).
Placental bikunin is a serine proteinase inhibitor containing two
Kunitz domains (Delaria et al., J. Biol. Chem. 272:12209-12214,
1997). Individual Kunitz domains of bikunin have been expressed and
shown to be potent inhibitors of trypsin, chymotrypsin, plasmin,
factor XIa, and tissue and plasma kallikrein (Delaria et al.,
ibid.).
[0007] Known Kunitz-type inhibitors lack specificity and may have
low potency. Lack of specificity can result in undesirable side
effects, such as nephrotoxicity that occurs after repeated
injections of high doses of aprotinin. These limitations may be
overcome by preparing isolated Kunitz domains, which may have fewer
side effects than traditional anticoagulants. Hence, there is a
need in the art for additional Kunitz-type proteinase
inhibitors.
BRIEF DESCRIPTION OF THE DRAWING
[0008] The attached drawing is a Hopp/Woods hydrophilicity profile
of the zkun10 protein sequence shown in SEQ ID NO:2. The profile is
based on a sliding six-residue window. Buried G, S, and T residues
and exposed H, Y, and W residues were ignored. These residues are
indicated in the figure by lower case letters.
DETAILED DESCRIPTION OF THE INVENTION
[0009] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms:
[0010] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification or detection of the second polypeptide or
provide sites for attachment of the second polypeptide to a
substrate. In principal, any peptide or protein for which an
antibody or other specific binding agent is available can be used
as an affinity tag. Affinity tags include a poly-histidine tract,
protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al.,
Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith and
Johnson, Gene 67:31, 1988), Glu-Glu affinity tag
(Glu-Tyr-Met-Pro-Met-Glu; SEQ ID NO:6) (Grussenmeyer et al., Proc.
Natl. Acad. Sci. USA 82:7952-4, 1985), substance P, Flag.TM.
peptide (Hopp et al., Biotechnology 6:1204-10, 1988), streptavidin
binding peptide, or other antigenic epitope or binding domain. See,
in general, Ford et al., Protein Expression and Purification 2:
95-107, 1991. DNAs encoding affinity tags are available from
commercial suppliers (e.g., Pharmacia Biotech, Piscataway,
N.J.).
[0011] The term "allelic variant" is used herein to denote any of
two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in phenotypic polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered amino acid
sequence. The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
[0012] The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides. Where the context
allows, these terms are used with reference to a particular
sequence or portion of a polypeptide to denote proximity or
relative position. For example, a certain sequence positioned
carboxyl-terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus of the
complete polypeptide.
[0013] A "complement" of a polynucleotide molecule is a
polynucleotide molecule having a complementary base sequence and
reverse orientation as compared to a reference sequence. For
example, the sequence 5' ATGCACGGG 3' is complementary to 5'
CCCGTGCAT 3'.
[0014] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons (as
compared to a reference polynucleotide molecule that encodes a
polypeptide). Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
[0015] A "DNA segment" is a portion of a larger DNA molecule having
specified attributes. For example, a DNA segment encoding a
specified polypeptide is a portion of a longer DNA molecule, such
as a plasmid or plasmid fragment, that, when read from the 5' to
the 3' direction, encodes the sequence of amino acids of the
specified polypeptide.
[0016] The term "expression vector" is used to denote a DNA
molecule, linear or circular, that comprises a segment encoding a
polypeptide of interest operably linked to additional segments that
provide for its transcription. Such additional segments include
promoter and terminator sequences, and may also include one or more
origins of replication, one or more selectable markers, an
enhancer, a polyadenylation signal, etc. Expression vectors are
generally derived from plasmid or viral DNA, or may contain
elements of both.
[0017] The term "isolated", when applied to a polynucleotide,
denotes that the polynucleotide has been removed from its natural
genetic milieu and is thus free of other extraneous or unwanted
coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions such as promoters and
terminators. The identification of associated regions will be
evident to one of ordinary skill in the art (see for example, Dynan
and Tijan, Nature 316:774-78, 1985).
[0018] An "isolated" polypeptide or protein is a polypeptide or
protein that is found in a condition other than its native
environment, such as apart from blood and animal tissue. In a
preferred form, the isolated polypeptide is substantially free of
other polypeptides, particularly other polypeptides of animal
origin. It is preferred to provide the polypeptides in a highly
purified form, i.e. greater than 95% pure, more preferably greater
than 99% pure. When used in this context, the term "isolated" does
not exclude the presence of the same polypeptide in alternative
physical forms, such as dimers or alternatively glycosylated or
derivatized forms.
[0019] The term "operably linked", when referring to DNA segments,
indicates that the segments are arranged so that they function in
concert for their intended purposes, e.g., transcription initiates
in the promoter and proceeds through the coding segment to the
terminator.
[0020] The term "ortholog" denotes a polypeptide or protein
obtained from one species that is the functional counterpart of a
polypeptide or protein from a different species. Sequence
differences among orthologs are the result of speciation.
[0021] A "polynucleotide" is a single- or double-stranded polymer
of deoxyribonucleotide or ribonucleotide bases read from the 5' to
the 3' end. Polynucleotides include RNA and DNA, and may be
isolated from natural sources, synthesized in vitro, or prepared
from a combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides ("nt"), or kilobases ("kb"). Where the context allows,
the latter two terms may describe polynucleotides that are
single-stranded or double-stranded. When these terms are applied to
double-stranded molecules they are used to denote overall length
and will be understood to be equivalent to the term "base pairs".
It will be recognized by those skilled in the art that the two
strands of a double-stranded polynucleotide may differ slightly in
length and that the ends thereof may be staggered as a result of
enzymatic cleavage; thus all nucleotides within a double-stranded
polynucleotide molecule may not be paired. Such unpaired ends will
in general not exceed 20 nt in length.
[0022] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides".
[0023] The term "promoter" is used herein for its art-recognized
meaning to denote a portion of a gene containing DNA sequences that
provide for the binding of RNA polymerase and initiation of
transcription. Promoter sequences are commonly, but not always,
found in the 5' non-coding regions of genes.
[0024] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0025] The term "secretory signal sequence" denotes a DNA sequence
that encodes a polypeptide (a "secretory peptide") that, as a
component of a larger polypeptide, directs the larger polypeptide
through a secretory pathway of a cell in which it is synthesized.
The larger polypeptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway.
[0026] The term "splice variant" is used herein to denote
alternative forms of RNA transcribed from a gene. Splice variation
arises naturally through use of alternative splicing sites within a
transcribed RNA molecule, or less commonly between separately
transcribed RNA molecules, and may result in several mRNAs
transcribed from the same gene. Splice variants may encode
polypeptides having altered amino acid sequence. The term splice
variant is also used herein to denote a protein encoded by a splice
variant of an mRNA transcribed from a gene.
[0027] Molecular weights and lengths of polymers determined by
imprecise analytical methods (e.g., gel electrophoresis) will be
understood to be approximate values. When such a value is expressed
as "about" X or "approximately" X, the stated value of X will be
understood to be accurate to .+-.10%.
[0028] All references cited herein are incorporated by reference in
their entirety.
[0029] The present invention provides, in part, novel serine
proteinases comprising a Kunitz domain. This Kunitz domain,
including sequence variants thereof and proteins containing it, is
referred to herein as "zkun10 ". The zkun10 polypeptide sequence
shown in SEQ ID NO:2 comprises this Kunitz domain, which is bounded
at the amino and carboxyl termini by cysteine residues at positions
57 and 107, respectively.
[0030] Zkun10 has been found to contain at least six exons, with
the kunitz domain encoded by nucleotide 1081 to 1233 of SEQ ID NO:
5. The structure as shown in SEQ ID NO: 6 describes a genomic
sequence wherein exon 1 comprises nucleotides 1-91; exon 2
comprises nucleotides 92-328; exon 3 comprises nucleotides 329-751;
exon 4 comprises nucleotides 752-845; exon 5 comprises nucleotides
846-1075; and exon 6 comprises nucleotides 1076-1747. The entire
kunitz domain resides within exon 6, and is represented in SEQ ID
NO: 5 from nucleotide 1081 to 1233.
[0031] While the sequence begins with a Met (at residue 1 of SEQ ID
NO: 6) it is likely that there is additional sequence at the
N-terminus of the polypeptide that comprises a signal sequence. The
von Willebrand factor domain is shown in SEQ ID NO: 6 as amino acid
residues 52 (Asp) to 211 (Leu), and the Kunitz domain is shown as
amino acid residues 361 (Cys) to 411 (Cys).
[0032] Human alpha 3 type VI collagen is a complex protein that
comprises from N-terminus to C-terminus, six von Willebrand
domains, a fibronectin III domain and a single kunitz domain. Human
alpha 3 type VI collagen also includes globular domains, which with
alpha 1 and alpha 2 chains assemble to form collagen type VI
(Lamande et al., J. Biol. Chem. 273:7423-7430, 1998.) The monomer
polypeptides form dimeric, and then tetrameric proteins, which
finally result in the formation of microfibrils. Human alpha 3 type
VI collagen is found in the subendothelium where it associated with
von Willebrand factor and may possibly serve as anchor for
interconnecting collagen fibers (Kehrel, Seminars in Thrombosis and
Hemostasis, 21:123-129,1995.)
[0033] zkun10 has approximately 27% homology with the von
Willebrand domain of human alpha 3 type VI collagen. The von
Willebrand domain is N-terminal to the kunitz domain. Von
Willebrand factor is a large plasma glycoprotein, which plays
essential roles in hemostasis (see, for example, Ruggeri, J. Clin.
Invest. 99:559 (1997)). The von Willebrand factor precursor
includes 13 domains that are multiples of domains A to D. The A
domains mediate key macromolecular interactions by von Willebrand
factor, and A domain mutations are associated with bleeding
disorders.
[0034] The von Willebrand factor type A domain is a characteristic
of a protein superfamily, and occurs in complement factors,
integrins, collagen, and other extracellular proteins (see, for
example, Colombatti et al., Matrix 13:297 (1993), and Bork and
Rhode, Biochem. J. 279:908 (1991)). Proteins comprising these type
A domains participate in a wide variety of biological processes,
including cell adhesion, cell migration, and signal transduction
(Jenkins et al., Blood 91:2032 (1998)). Certain proteins that
contain one or more copies of the type A domain take part in host
defense mechanisms, such as immune response and inflammation (see,
for example, Celikel et al., Nature Structural Biology 5:189
(1998)).
[0035] Zkun10 has 51% residue identity with the 51-residue kunitz
domain in human alpha 3 type VI collagen. The structure of the
latter domain has been solved by X-ray crystallography and by NMR
(Arnoux et al., J. Mol. Biol. 246:609-617, 1995; Sorensen et al.,
Biochemistry 36:10439-10450, 1997). An alignment of zkun10 and the
collagen Kunitz domain (see the drawing) can be combined with a
homology model of zkun10 based on the X-ray structure to predict
the function of certain residues in zkun10. Referring to SEQ ID
NO:2, disulfide bonds are predicted to be formed by paired cysteine
residues Cys57 -Cys107; Cys66 -Cys90; and Cys82 -Cys103. An
unpaired Cysteine is found at residue 87, and is buried in the
hydrophobic coil of the molecule. While a similar cysteine
conformation is not unusual in some proteins, e.g. the fibroblast
growth family proteins, it is not common to the kunitz protein
family. Therefore, the residue at Cys87 of SEQ ID NO: 2, may be
substituted as defined by the limitations of corresponding residue
31 of SEQ ID NO: 4. The protease binding loop (P3-P4') is expected
to comprise residues 65-71 of SEQ ID NO:2
(Glu-Cys-Gln-Asp-His-Thr-Leu), with the P1 residue being Gln67, and
the P1' residue being Asp68.
[0036] Amino acid substitutions can be made within the zkun10
sequence so long as the conserved cysteine residues are retained
and the higher order structure is not disrupted. It is preferred to
make substitutions within the zkun10 Kunitz domain by reference to
the sequences of other Kunitz domains. SEQ ID NO:4 is a generalized
Kunitz domain sequence that shows allowable amino acid
substitutions based on such an alignment. However, mutants can be
made that would purposely alter binding specificity and inhibition
profiles. The 51-residue sequence shown in SEQ ID NO:4 conforms to
the pattern:
C-X(8)-C-X(15)-C-X(7)-C-X(12)-C-X(3)-C
[0037] wherein C denotes cysteine; X is any naturally occurring
amino acid residue, subject to the limitations set forth in the
attached Sequence Listing for SEQ ID NO:4; and the numerals
indicate the number of such variable residues. The second cysteine
residue is in the P2 position.
[0038] Within the present invention up to 20% of the amino acid
residues in the zkun10 Kunitz domain (residues 57 through 107 of
SEQ ID NO:2) can be replaced with other amino acid residues,
subject to the limitation that the resulting substituted sequence
is one of the sequences disclosed in SEQ ID NO:4. The present
invention thus provides a family of proteins comprising a sequence
of amino acid residues as shown in SEQ ID NO:4, wherein the
sequence is at least 80% identical to residues 57 through 107 of
SEQ ID NO:2. In other embodiments of the present invention, the
proteins of the present invention comprise such a sequence that is
at least 85%, at least 90%, and at least 95%, 96%, 97%, 98%, or 99%
identical to residues 57 through 107 of SEQ ID NO:2.
[0039] In other embodiments, the present invention comprises the
entire sequence as shown in SEQ ID NOS: 5 and 6. The Kunitz domain
resides within this sequence as well (amino acid residues 361-411
of SEQ ID NO: 6), and therefore, substitutions will be limited
within that domain to those described above, and shown in the
respective locations of SEQ ID NO: 4. With regards to the larger
collagen type polypeptides and proteins, certain embodiments of the
present invention, the polypeptides and proteins of the present
invention comprise such a sequence that is at least 85%, at least
90%, and at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID
NO:6, with the limitations shown in SEQ ID NO: 4 for corresponding
regions.
[0040] Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio.
48:603-616, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915-10919, 1992. Briefly, two amino acid sequences are
aligned to optimize the alignment scores using a gap opening
penalty of 10, a gap extension penalty of 1, and the "BLOSUM62"
scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 1
(amino acids are indicated by the standard one-letter codes). The
percent identity is then calculated as: 1 Total number of identical
matches [ length of the longer sequence plus the number of gaps
introduced into the longer sequence in order to align the two
sequences ] .times. 100
1 TABLE 1 A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 5 N -2 0
6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0
-2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3
-4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1
-3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3
-3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1
-1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1
-1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3
-2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3
-3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
[0041] The level of identity between amino acid sequences can be
determined using the "FASTA" similarity search algorithm disclosed
by Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988)
and by Pearson (Meth. Enzymol. 183:63, 1990). Briefly, FASTA first
characterizes sequence similarity by identifying regions shared by
the query sequence (e.g., SEQ ID NO:2) and a test sequence that
have either the highest density of identities (if the ktup variable
is 1) or pairs of identities (if ktup=2), without considering
conservative amino acid substitutions, insertions, or deletions.
The ten regions with the highest density of identities are then
rescored by comparing the similarity of all paired amino acids
using an amino acid substitution matrix, and the ends of the
regions are "trimmed" to include only those residues that
contribute to the highest score. If there are several regions with
scores greater than the "cutoff" value (calculated by a
predetermined formula based upon the length of the sequence and the
ktup value), then the trimmed initial regions are examined to
determine whether the regions can be joined to form an approximate
alignment with gaps. Finally, the highest scoring regions of the
two amino acid sequences are aligned using a modification of the
Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.
Biol. 48:444, 1970; Sellers, SIAM J. Appl. Math. 26:787, 1974),
which allows for amino acid insertions and deletions. Preferred
parameters for FASTA analysis are: ktup=1, gap opening penalty=10,
gap extension penalty=1, and substitution matrix=BLOSUM62. These
parameters can be introduced into a FASTA program by modifying the
scoring matrix file ("SMATRIX"), as explained in Appendix 2 of
Pearson, 1990 (ibid.).
[0042] FASTA can also be used to determine the sequence identity of
nucleic acid molecules using a ratio as disclosed above. For
nucleotide sequence comparisons, the ktup value can range between
one to six, preferably from three to six, most preferably three,
with other parameters set as default.
[0043] The proteins of the present invention can also comprise
non-naturally occurring amino acid residues. Non-naturally
occurring amino acids include, without limitation,
trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
trans-4-hydroxyproline, N-methylglycine, allo-threonine,
methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,
nitroglutamine, homoglutamine, pipecolic acid, thiazolidine
carboxylic acid, dehydroproline, 3- and 4-methylproline,
3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
Several methods are known in the art for incorporating
non-naturally occurring amino acid residues into proteins. For
example, an in vitro system can be employed wherein nonsense
mutations are suppressed using chemically aminoacylated suppressor
tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA
are known in the art. Transcription and translation of plasmids
containing nonsense mutations is carried out in a cell-free system
comprising an E. coli S30 extract and commercially available
enzymes and other reagents. Proteins are purified by
chromatography. See, for example, Robertson et al., J. Am. Chem.
Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991;
Chung et al., Science 259:806-9, 1993; and Chung et al., Proc.
Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method,
translation is carried out in Xenopus oocytes by microinjection of
mutated mRNA and chemically aminoacylated suppressor tRNAs
(Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Within a third
method, E. coli cells are cultured in the absence of a natural
amino acid that is to be replaced (e.g., phenylalanine) and in the
presence of the desired non-naturally occurring amino acid(s)
(e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine,
or 4-fluorophenylalanine). The non-naturally occurring amino acid
is incorporated into the protein in place of its natural
counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally
occurring amino acid residues can be converted to non-naturally
occurring species by in vitro chemical modification. Chemical
modification can be combined with site-directed mutagenesis to
further expand the range of substitutions (Wynn and Richards,
Protein Sci. 2:395-403, 1993).
[0044] Amino acid sequence changes described herein are made in
zkun10 polypeptides so as to minimize disruption of higher order
structure essential to biological activity. Amino acid residues
that are critical to maintaining structural integrity can be
determined. As shown in SEQ ID NO: 4 specific residues that will be
more or less tolerant of change and maintain the overall tertiary
structure of the molecule have been described. Methods for
analyzing sequence structure include, but are not limited to,
alignment of multiple sequences with high amino acid or nucleotide
identity, secondary structure propensities, binary patterns,
complementary packing, and buried polar interactions (Barton,
Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al.,
Current Opin. Struct. Biol. 6:3-10, 1996). In general,
determination of structure will be accompanied by evaluation of
activity of modified molecules. For example, changes in amino acid
residues will be made so as not to disrupt the protease binding
loop structure of the protein family. The effects of amino acid
sequence changes can be predicted by, for example, computer
modeling using available software (e.g., the Insight II.RTM. viewer
and homology modeling tools; MSI, San Diego, Calif.) or determined
by analysis of crystal structure (see, e.g., Lapthorn et al, Nature
369:455-461, 1994; Lapthorn et al., Nat. Struct. Biol. 2:266-268,
1995). Protein folding can be measured by circular dichroism (CD).
Measuring and comparing the CD spectra generated by a modified
molecule and standard molecule are routine in the art (Johnson,
Proteins 7:205-214, 1990). Crystallography is another well known
and accepted method for analyzing folding and structure. Nuclear
magnetic resonance (NMR), digestive peptide mapping and epitope
mapping are other known methods for analyzing folding and
structural similarities between proteins and polypeptides (Schaanan
et al., Science 257:961-964, 1992). Mass spectrometry and chemical
modification using reduction and alkylation can be used to identify
cysteine residues that are associated with disulfide bonds or are
free of such associations (Bean et al., Anal. Biochem. 201:216-226,
1992; Gray, Protein Sci. 2:1732-1748, 1993; and Patterson et al.,
Anal. Chem. 66:3727-3732, 1994). Alterations in disulfide bonding
will be expected to affect protein folding. These techniques can be
employed individually or in combination to analyze and compare the
structural features that affect folding of a variant protein or
polypeptide to a standard molecule to determine whether such
modifications would be significant.
[0045] Essential amino acids in the polypeptides of the present
invention can be identified experimentally according to procedures
known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis (Cunningham and Wells, Science 244,
1081-1085, 1989; Bass et al., Proc. Natl. Acad. Sci. USA
88:4498-4502, 1991). In the latter technique, single alanine
mutations are introduced at every residue in the molecule, and the
resultant mutant molecules are tested for biological activity as
disclosed below to identify amino acid residues that are critical
to the activity of the molecule.
[0046] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer (Science 241:53-57, 1988) or
Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991; Ladner
et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO
92/06204) and region-directed mutagenesis (Derbyshire et al., Gene
46:145, 1986; Ner et al., DNA 7:127, 1988).
[0047] Variants of the disclosed zkun10 DNA and polypeptide
sequences can be generated through DNA shuffling as disclosed by
Stemmer, Nature 370:389-391, 1994 and Stemmer, Proc. Natl. Acad.
Sci. USA 91:10747-10751, 1994. Briefly, variant genes are generated
by in vitro homologous recombination by random fragmentation of a
parent gene followed by reassembly using PCR, resulting in randomly
introduced point mutations. This technique can be modified by using
a family of parent genes, such as allelic variants or genes from
different species, to introduce additional variability into the
process. Selection or screening for the desired activity, followed
by additional iterations of mutagenesis and assay provides for
rapid "evolution" of sequences by selecting for desirable mutations
while simultaneously selecting against detrimental changes.
[0048] In many cases, the structure of the final polypeptide
product will result from processing of the nascent polypeptide
chain by the host cell, thus the final sequence of a zkun10
polypeptide produced by a host cell will not always correspond to
the full sequence encoded by the expressed polynucleotide. For
example, expressing the complete zkun10 sequence in a cultured
mammalian cell is expected to result in removal of at least the
secretory peptide, while the same polypeptide produced in a
prokaryotic host would not be expected to be cleaved. Differential
processing of individual chains may result in heterogeneity of
expressed polypeptides.
[0049] Additional polypeptides may be joined to the amino and/or
carboxyl termini of the zkun10 Kunitz domain (residues 57-107 of
SEQ ID NO:2) or a derivative of the zkun10 Kunitz domain as
disclosed above. Amino and carboxyl extensions of the zkun10 Kunitz
domain will be selected so as not to destroy or mask the
proteinase-inhibiting activity of the protein by, for example,
burying the Kunitz domain within the interior of the protein. There
is a consequent preference for shorter extensions, typically 10-15
residues in length, preferably not exceeding 8 residues in length.
There is considerable latitude in the permissible sequence of these
extensions, although it is preferred to avoid the addition of
cysteine residues in close proximity to the the Kunitz domain
itself. For example, a zkun10 protein can comprise residues 57-107
of SEQ ID NO:2 with amino- and carboxyl-terminal dipeptides,
wherein the individual amino acid residues of the dipeptides are
any amino acid residue except cysteine. Of particular interest are
extensions derived from other members of the Kunitz family proteins
and collagen family. The nucleotide sequences and encoded
polypeptide domains of the present invention are particularly
suited for construction of chimeric molecules comprising a portion
of zkun10 and portions from one or more other proteins containing
Kunitz domains.
[0050] Other amino- and carboxyl-terminal extensions that can be
included in the proteins of the present invention include, for
example, an amino-terminal methionine residue, a small linker
peptide of up to about 20-25 residues, or an affinity tag as
disclosed above. A protein comprising such an extension may further
comprise a polypeptide linker and/or a proteolytic cleavage site
between the zkun10 portion and the affinity tag. Preferred cleavage
sites include thrombin cleavage sites and factor Xa cleavage sites.
For example, a zkun10 polypeptide of 50 amino acid residues can be
expressed as a fusion comprising, from amino terminus to carboxyl
terminus: maltose binding protein (approximately 370
residues)--polyhistidine (6 residues)--thrombin cleavage site
(Leu-Val-Pro-Arg; SEQ ID NO:5)--zkun10, resulting in a polypeptide
of approximately 430 residues. In a second example, a zkun10
polypeptide of 50 residues can be fused to E. coli
.beta.-galactosidase (1,021 residues; see Casadaban et al., J.
Bacteriol. 143:971-980, 1980), a 10-residue spacer, and a 4-residue
factor Xa cleavage site to yield a polypeptide of 1,085 residues.
Linker peptides and affinity tags provide for additional functions,
such as binding to substrates, antibodies, binding proteins, and
the like, and facilitate purification, detection, and delivery of
zkun10 proteins. In another example, a zkun10 Kunitz domain can be
expressed as a secreted protein comprising a carboxyl-terminal
receptor transmembrane domain, permitting the Kunitz domain to be
displayed on the surface of a cell. To span the lipid bilayer of
the cell membrane, a minimum of about 20 amino acids are required
in the transmembrane domain; these should predominantly be
hydrophobic amino acids. The Kunitz domain can be separated from
the transmembrane domain by a spacer polypeptide, and can be
contained within an extended polypeptide comprising a
carboxyl-terminal transmembrane domain--spacer polypeptide--Kunitz
domain--amino-terminal polypeptide. Many receptor transmembrane
domains and polynucleotides encoding them are known in the art. The
spacer polypeptide will generally be at least about 50 amino acid
residues in length, up to 200-300 or more residues. The amino
terminal polypeptide may be up to 300 or more residues in
length.
[0051] The present invention also provides polypeptide fragments or
peptides comprising an epitope-bearing portion of a Zkun10
polypeptide described herein. Such fragments or peptides may
comprise an "immunogenic epitope," which is a part of a protein
that elicits an antibody response when the entire protein is used
as an immunogen. Immunogenic epitope-bearing peptides can be
identified using standard methods (see, for example, Geysen et al.,
Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).
[0052] In contrast, polypeptide fragments or peptides may comprise
an "antigenic epitope," which is a region of a protein molecule to
which an antibody can specifically bind. Certain epitopes consist
of a linear or contiguous stretch of amino acids, and the
antigenicity of such an epitope is not disrupted by denaturing
agents. It is known in the art that relatively short synthetic
peptides that can mimic epitopes of a protein can be used to
stimulate the production of antibodies against the protein (see,
for example, Sutcliffe et al., Science 219:660 (1983)). Antibodies
that recognize short linear epitopes are particularly useful in
analytic and diagnostic applications that use denatured protein,
such as Western analysis, or in the analysis of fixed cells or
tissue samples. Antibodies to linear epitopes are also useful for
detecting fragments of Zkun10, such as might occur in body fluids
or culture media. Accordingly, antigenic epitope-bearing peptides
and polypeptides of the present invention are useful to raise
antibodies that bind with the polypeptides described herein.
[0053] Antigenic epitope-bearing peptides and polypeptides can
contain at least four to ten amino acids, at least ten to fifteen
amino acids, or about 15 to about 30 amino acids of SEQ ID NO:2.
Such epitope-bearing peptides and polypeptides can be produced by
fragmenting a Zkun10 polypeptide, or by chemical peptide synthesis,
as described herein. Moreover, epitopes can be selected by phage
display of random peptide libraries (see, for example, Lane and
Stephen, Curr. Opin. Immunol 5:268 (1993), and Cortese et al.,
Curr. Opin. Biotechnol. 7:616 (1996)). Standard methods for
identifying epitopes and producing antibodies from small peptides
that comprise an epitope are described, for example, by Mole,
"Epitope Mapping," in Methods in Molecular Biology, Vol. 10, Manson
(ed.), pages 105-116 (The Humana Press, Inc. 1992), Price,
"Production and Characterization of Synthetic Peptide-Derived
Antibodies," in Monoclonal Antibodies: Production, Engineering, and
Clinical Application, Ritter and Ladyman (eds.), pages 60-84
(Cambridge University Press 1995), and Coligan et al. (eds.),
Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages
9.4.1-9.4.11 (John Wiley & Sons 1997).
[0054] In particular, fragments of interest include those
containing the kunitz domain of amino acid residues 361-411 of SEQ
ID NO: 6, and amino acid residues 313-415 which encompasses the
kunitz domain and a sequence with similarity to a high affinity
binding processing site found in other proteinase inhibitor
proteins such as TFPI-2.
[0055] The present invention thus provides a series of hybrid
molecules in which a segment comprising one or more of the domains
of zkun10 is fused to another polypeptide. Fusion is preferably
done by splicing at the DNA level to allow expression of chimeric
molecules in recombinant production systems. The resultant
molecules are then assayed for such properties as improved
solubility, improved stability, prolonged clearance half-life,
improved expression and secretion levels, and pharmacodynamics.
Such hybrid molecules may further comprise additional amino acid
residues (e.g. a polypeptide linker) between the component proteins
or polypeptides. The present invention further provides a variety
of other polypeptide fusions (and related multimeric proteins
comprising one or more polypeptide fusions). For example, a zkun10
polypeptide can be prepared as a fusion to a dimerizing protein as
disclosed in U.S. Pat. Nos. 5,155,027 and 5,567,584. Preferred
dimerizing proteins in this regard include immunoglobulin constant
region domains. Immunoglobulin-zkun10 polypeptide fusions can be
expressed in genetically engineered cells (to produce a variety of
multimeric zkun10 analogs). Auxiliary domains can be fused to
zkun10 polypeptides to target them to specific cells, tissues, or
macromolecules. For example, a zkun10 polypeptide or protein could
be targeted to a predetermined cell type by fusing a zkun10
polypeptide to a ligand that specifically binds to a receptor on
the surface of that target cell. In this way, polypeptides and
proteins can be targeted for therapeutic or diagnostic purposes. A
zkun10 polypeptide can be fused to two or more moieties, such as an
affinity tag for purification and a targeting domain. Polypeptide
fusions can also comprise one or more cleavage sites, particularly
between domains. See, Tuan et al., Connective Tissue Research
34:1-9, 1996.
[0056] Fusion proteins can be prepared by methods known to those
skilled in the art by preparing each component of the fusion
protein and chemically conjugating them. Alternatively, a
polynucleotide encoding both components of the fusion protein in
the proper reading frame can be generated using known techniques
and expressed by the methods described herein. For example, part or
all of a collagen or Kunitz conferring a biological function may be
swapped between zkun10 of the present invention with the
functionally equivalent domains from another family member, such as
Type 6 collagen, TFPI or TFPI-2. Such components include, but are
not limited to, the secretory signal sequence; globular domains,
Kunitz domains, helical domains, and von Willebrand domains. Such
fusion proteins would be expected to have a biological functional
profile that is the same or similar to polypeptides of the present
invention or other known serine protease inhibitor family proteins,
depending on the fusion constructed. Moreover, such fusion proteins
may exhibit other properties as disclosed herein.
[0057] Standard molecular biological and cloning techniques can be
used to swap the equivalent domains between the zkun10 polypeptide
and those polypeptides to which they are fused. Generally, a DNA
segment that encodes a domain of interest, e.g., zkun10 Kunitz, or
other domains described herein, is operably linked in frame to at
least one other DNA segment encoding an additional polypeptide, and
inserted into an appropriate expression vector, as described
herein. Generally DNA constructs are made such that the several DNA
segments that encode the corresponding regions of a polypeptide are
operably linked in frame to make a single construct that encodes
the entire fusion protein, or a functional portion thereof. For
example, a DNA construct would encode from N-terminus to C-terminus
a fusion protein comprising a signal polypeptide followed by a
collagen domain fusion protein containing one or more von
Willebrand domains, followed by one or more Kunitz domains. Such
fusion proteins can be expressed, isolated, and assayed for
activity as described herein.
[0058] Also disclosed herein are polynucleotide molecules,
including DNA and RNA molecules, encoding zkun10 proteins. These
polynucleotides include the sense strand; the anti-sense strand;
and the DNA as double-stranded, having both the sense and
anti-sense strand annealed together by their respective hydrogen
bonds. A representative DNA sequence encoding a zkun10 protein is
set forth in SEQ ID NO:1. DNA sequences encoding other zkun10
proteins can be readily generated by those of ordinary skill in the
art based on the genetic code. Counterpart RNA sequences can be
generated by substitution of U for T. Polynucleotides encoding
zkun10 proteins and complementary polynucleotides are useful in the
production of zkun10 proteins and for diagnostic and investigatory
purposes.
[0059] Those skilled in the art will readily recognize that, in
view of the degeneracy of the genetic code, considerable sequence
variation is possible among these polynucleotide molecules. SEQ ID
NO:3 is a degenerate DNA sequence that encompasses all DNAs that
encode the zkun10 polypeptide of SEQ ID NO:2. Those skilled in the
art will recognize that the degenerate sequence of SEQ ID NO:3 also
provides all RNA sequences encoding SEQ ID NO:2 by substituting U
for T. Thus, zkun10 polypeptide-encoding polynucleotides comprising
nucleotide 158 to nucleotide 333 of SEQ ID NO:3 and their
respective RNA equivalents are contemplated by the present
invention. Table 2 sets forth the one-letter codes used within SEQ
ID NO:3 to denote degenerate nucleotide positions. "Resolutions"
are the nucleotides denoted by a code letter. "Complement"
indicates the code for the complementary nucleotide(s). For
example, the code Y denotes either C or T, and its complement R
denotes A or G, A being complementary to T, and G being
complementary to C.
2TABLE 2 Nucleotide Resolution Nucleotide Complement A A T T C C G
G G G C C T T A A R A.vertline.G Y C.vertline.T Y C.vertline.T R
A.vertline.G M A.vertline.C K G.vertline.T K G.vertline.T M
A.vertline.C S G.vertline.G S G.vertline.G W A.vertline.T W
A.vertline.T H A.vertline.C.vertline.T D A.vertline.G.vertline.T B
C.vertline.G.vertline.T V A.vertline.C.vertline.G V
A.vertline.C.vertline.G B C.vertline.G.vertline.T D
A.vertline.G.vertline.T H A.vertline.C.vertline.T N
A.vertline.C.vertline.G.vertlin- e.T N
A.vertline.C.vertline.G.vertline.T
[0060] The degenerate codons used in SEQ ID NO:4, encompassing all
possible codons for a given amino acid, are set forth in Table
3.
3TABLE 3 One Amino Letter Degenerate Acid Code Codons Codon Cys C
TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT
ACN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA
GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG
GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG
CGT MGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L
CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GIG GTT GTN Phe F TTC TTT
TTY Tyr Y TAC TAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRR
Asn.vertline.Asp B RAY Glu.vertline.Gln Z SAR Any X NNN
[0061] One of ordinary skill in the art will appreciate that some
ambiguity is introduced in determining a degenerate codon,
representative of all possible codons encoding each amino acid. For
example, the degenerate codon for serine (WSN) can, in some
circumstances, encode arginine (AGR), and the degenerate codon for
arginine (MGN) can, in some circumstances, encode serine (AGY). A
similar relationship exists between codons encoding phenylalanine
and leucine. Thus, some polynucleotides encompassed by the
degenerate sequence may encode variant amino acid sequences, but
one of ordinary skill in the art can easily identify such variant
sequences by reference to the amino acid sequences shown in SEQ ID
NO:2. Variant sequences can be readily tested for functionality as
described herein.
[0062] One of ordinary skill in the art will also appreciate that
different species can exhibit preferential codon usage. See, in
general, Grantham et al., Nuc. Acids Res. 8:1893-912, 1980; Haas et
al. Curr. Biol. 6:315-24, 1996; Wain-Hobson et al., Gene 13:355-64,
1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids
Res. 14:3075-87, 1986; and Ikemura, J. Mol. Biol. 158:573-97, 1982.
"Preferential codon usage" is a term of art referring to the bias
in codon usage within the genomes of certain species, whereby
certain protein translation codons are more frequently used, thus
favoring one or a few representatives of the possible codons
encoding each amino acid (see Table 3). For example, the amino acid
threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in
mammalian cells ACC is the most commonly used codon. In other
species, for example, insect cells, yeast, viruses or bacteria,
different Thr codons may be preferred. Preferred codons for a
particular species can be introduced into the polynucleotides of
the present invention by a variety of methods known in the art.
Introduction of preferred codon sequences into recombinant DNA can,
for example, enhance production of the protein by making protein
translation more efficient within a particular cell type or
species. Therefore, the degenerate codon sequence disclosed in SEQ
ID NO:4 serves as a template for optimizing expression of
polynucleotides in various cell types and species commonly used in
the art and disclosed herein. Sequences containing preferred codons
can be tested and optimized for expression in various host cell
species, and tested for functionality as disclosed herein.
[0063] Within certain embodiments of the invention the isolated
polynucleotides will hybridize to similar sized regions of SEQ ID
NO:1 or a sequence complementary thereto under stringent
conditions. In general, stringent conditions are selected to be
about 5.degree. C. lower than the thermal melting point (T.sub.m)
for the specific sequence at a defined ionic strength and pH. The
T.sub.m is the temperature (under defined ionic strength and pH) at
which 50% of the target sequence hybridizes to a perfectly matched
probe. Typical stringent conditions are those in which the salt
concentration is up to about 0.03 M at pH 7 and the temperature is
at least about 60.degree. C.
[0064] As previously noted, the isolated polynucleotides of the
present invention include DNA and RNA. Methods for preparing DNA
and RNA are well known in the art. In general, RNA is isolated from
a tissue or cell that produces large amounts of zkun10 RNA. Total
RNA can be prepared using guanidine HCl extraction followed by
isolation by centrifugation in a CsCl gradient (Chirgwin et al.,
Biochemistry 18:52-94, 1979). Poly (A).sup.+ RNA is prepared from
total RNA using the method of Aviv and Leder (Proc. Natl. Acad.
Sci. USA 69:1408-1412, 1972). Complementary DNA (cDNA) is prepared
from poly(A).sup.+ RNA using known methods. In the alternative,
genomic DNA can be isolated. Polynucleotides encoding zkun10
polypeptides are then identified and isolated by, for example,
hybridization or PCR.
[0065] Full-length clones encoding zkun10 can be obtained by
conventional cloning procedures. Complementary DNA (cDNA) clones
are preferred, although for some applications (e.g., expression in
transgenic animals) it may be preferable to use a genomic clone, or
to modify a cDNA clone to include at least one genomic intron.
Methods for preparing cDNA and genomic clones are well known and
within the level of ordinary skill in the art, and include the use
of the sequence disclosed herein, or parts thereof, for probing or
priming a library. Expression libraries can be probed with
antibodies to zkun10, receptor fragments, or other specific binding
partners.
[0066] Zkun10 polynucleotide sequences disclosed herein can also be
used as probes or primers to clone 5' non-coding regions of a
zkun10 gene. Promoter elements from a zkun10 gene can thus be used
to direct the expression of heterologous genes in, for example,
transgenic animals or patients treated with gene therapy. Cloning
of 5' flanking sequences also facilitates production of zkun10
proteins by "gene activation" as disclosed in U.S. Pat. No.
5,641,670. Briefly, expression of an endogenous zkun10 gene in a
cell is altered by introducing into the zkun10 locus a DNA
construct comprising at least a targeting sequence, a regulatory
sequence, an exon, and an unpaired splice donor site. The targeting
sequence is a zkun10 5' non-coding sequence that permits homologous
recombination of the construct with the endogenous zkun10 locus,
whereby the sequences within the construct become operably linked
with the endogenous zkun10 coding sequence. In this way, an
endogenous zkun10 promoter can be replaced or supplemented with
other regulatory sequences to provide enhanced, tissue-specific, or
otherwise regulated expression.
[0067] Those skilled in the art will recognize that the sequences
disclosed in SEQ ID NOS:1 and 2 represent a single allele of human
zkun10. Allelic variants of these sequences can be cloned by
probing cDNA or genomic libraries from different individuals
according to standard procedures.
[0068] The present invention further provides counterpart
polypeptides and polynucleotides from other species ("orthologs").
Of particular interest are zkun10 polypeptides from other mammalian
species, including murine, porcine, ovine, bovine, canine, feline,
equine, and other primate polypeptides. Orthologs of human zkun10
can be cloned using information and compositions provided by the
present invention in combination with conventional cloning
techniques. For example, a cDNA can be cloned using mRNA obtained
from a tissue or cell type that expresses zkun10 as disclosed
above. A library is then prepared from mRNA of a positive tissue or
cell line. A zkun10-encoding cDNA can then be isolated by a variety
of methods, such as by probing with a complete or partial human
cDNA or with one or more sets of degenerate probes based on the
disclosed sequence. A cDNA can also be cloned using the polymerase
chain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202), using
primers designed from the representative human zkun10 sequence
disclosed herein. Within an additional method, the cDNA library can
be used to transform or transfect host cells, and expression of the
cDNA of interest can be detected with an antibody to zkun10
polypeptide. Similar techniques can also be applied to the
isolation of genomic clones.
[0069] Nucleic acid molecules can be used to detect the expression
of a Zkun10 gene in a biological sample. Such probe molecules
include double-stranded nucleic acid molecules comprising the
nucleotide sequence of SEQ ID NO:1, or a fragment thereof, as well
as single-stranded nucleic acid molecules having the complement of
the nucleotide sequence of SEQ ID NO:1, or a portion thereof. As
used herein, the term "portion" refers to at least eight
nucleotides to at least 20 or more nucleotides. Probe molecules may
be DNA, RNA, oligonucleotides, and the like. Certain probes bind
with regions of a Zkun10 gene that have a low sequence similarity
to comparable regions in other serine protease inhibitors.
[0070] In a basic assay, a single-stranded probe molecule is
incubated with RNA, isolated from a biological sample, under
conditions of temperature and ionic strength that promote base
pairing between the probe and target Zkun10 RNA species. After
separating unbound probe from hybridized molecules, the amount of
hybrids is detected.
[0071] Well-established hybridization methods of RNA detection
include northern analysis and dot/slot blot hybridization (see, for
example, Ausubel (1995) at pages 4-1 to 4-27, and Wu et al. (eds.),
"Analysis of Gene Expression at the RNA Level," in Methods in Gene
Biotechnology, pages 225-239 (CRC Press, Inc. 1997)). Nucleic acid
probes can be detectably labeled with radioisotopes such as
.sup.32P or .sup.35S. Alternatively, Zkun10 RNA can be detected
with a nonradioactive hybridization method (see, for example, Isaac
(ed.), Protocols for Nucleic Acid Analysis by Nonradioactive Probes
(Humana Press, Inc. 1993)). Typically, nonradioactive detection is
achieved by enzymatic conversion of chromogenic or chemiluminescent
substrates. Illustrative nonradioactive moieties include biotin,
fluorescein, and digoxigenin.
[0072] Zkun10 oligonucleotide probes are also useful for in vivo
diagnosis. As an illustration, .sup.18F-labeled oligonucleotides
can be administered to a subject and visualized by positron
emission tomography (Tavitian et al., Nature Medicine 4:467
(1998)).
[0073] Numerous diagnostic procedures take advantage of the
polymerase chain reaction (PCR) to increase sensitivity of
detection methods. Standard techniques for performing PCR are
well-known (see, generally, Mathew (ed.), Protocols in Human
Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR
Protocols: Current Methods and Applications (Humana Press, Inc.
1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press,
Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols
(Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR
(Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis
(Humana Press, Inc. 1998)).
[0074] One variation of PCR for diagnostic assays is reverse
transcriptase-PCR (RT-PCR). In the RT-PCR technique, RNA is
isolated from a biological sample, reverse transcribed to cDNA, and
the cDNA is incubated with Zkun10 primers (see, for example, Wu et
al. (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR," in
Methods in Gene Biotechnology, pages 15-28 (CRC Press, Inc. 1997)).
PCR is then performed and the products are analyzed using standard
techniques.
[0075] As an illustration, RNA is isolated from biological sample
using, for example, the guanidinium-thiocyanate cell lysis
procedure described above. Alternatively, a solid-phase technique
can be used to isolate mRNA from a cell lysate. A reverse
transcription reaction can be primed with the isolated RNA using
random oligonucleotides, short homopolymers of dT, or Zkun10
anti-sense oligomers. Oligo-dT primers offer the advantage that
various mRNA nucleotide sequences are amplified that can provide
control target sequences. Zkun10 sequences are amplified by the
polymerase chain reaction using two flanking oligonucleotide
primers that are typically 20 bases in length.
[0076] PCR amplification products can be detected using a variety
of approaches. For example, PCR products can be fractionated by gel
electrophoresis, and visualized by ethidium bromide staining.
Alternatively, fractionated PCR products can be transferred to a
membrane, hybridized with a detectably-labeled Zkun10 probe, and
examined by autoradiography. Additional alternative approaches
include the use of digoxigenin-labeled deoxyribonucleic acid
triphosphates to provide chemiluminescence detection, and the
C-TRAK calorimetric assay.
[0077] Another approach for detection of Zkun10 expression is
cycling probe technology, in which a single-stranded DNA target
binds with an excess of DNA-RNA-DNA chimeric probe to form a
complex, the RNA portion is cleaved with RNAase II, and the
presence of cleaved chimeric probe is detected (see, for example,
Beggs et al., J. Clin. Microbiol. 34:2985 (1996), Bekkaoui et al.,
Biotechniques 20:240 (1996)). Alternative methods for detection of
Zknu10 sequences can utilize approaches such as nucleic acid
sequence-based amplification, cooperative amplification of
templates by cross-hybridization, and the ligase chain reaction
(see, for example, Marshall et al., U.S. Pat. No. 5,686,272 (1997),
Dyer et al., J. Virol. Methods 60:161 (1996), Ehricht et al., Eur.
J. Biochem. 243:358 (1997), and Chadwick et al., J. Virol. Methods
70:59 (1998)). Other standard methods are known to those of skill
in the art.
[0078] Zkun10 probes and primers can also be used to detect and to
localize Zkun10 gene expression in tissue samples. Methods for such
in situ hybridization are well-known to those of skill in the art
(see, for example, Choo (ed.), In Situ Hybridization Protocols
(Humana Press, Inc. 1994), Wu et al. (eds.), "Analysis of Cellular
DNA or Abundance of mRNA by Radioactive In Situ Hybridization
(RISH)," in Methods in Gene Biotechnology, pages 259-278 (CRC
Press, Inc. 1997), and Wu et al. (eds.), "Localization of DNA or
Abundance of mRNA by Fluorescence In Situ Hybridization (RISH)," in
Methods in Gene Biotechnology, pages 279-289 (CRC Press, Inc.
1997)). Various additional diagnostic approaches are well-known to
those of skill in the art (see, for example, Mathew (ed.),
Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),
Coleman and Tsongalis, Molecular Diagnostics (Humana Press, Inc.
1996), and Elles, Molecular Diagnosis of Genetic Diseases (Humana
Press, Inc., 1996)).
[0079] Zkun10 gene has been localized to 3q21.3 of the human
genome. Zkun10 nucleotide sequences can be used in linkage-based
testing for various diseases, and to determine whether a subject's
chromosomes contain a mutation in the Zkun10 gene. Detectable
chromosomal aberrations at the Zkun10 gene locus include, but are
not limited to, aneuploidy, gene copy number changes, insertions,
deletions, restriction site changes and rearrangements. Of
particular interest are genetic alterations that inactivate a
Zkun10 gene.
[0080] Aberrations associated with a Zkun10 locus can be detected
using nucleic acid molecules of the present invention by employing
molecular genetic techniques, such as restriction fragment length
polymorphism analysis, short tandem repeat analysis employing PCR
techniques, amplification-refractory mutation system analysis,
single-strand conformation polymorphism detection, RNase cleavage
methods, denaturing gradient gel electrophoresis,
fluorescence-assisted mismatch analysis, and other genetic analysis
techniques known in the art (see, for example, Mathew (ed.),
Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),
Marian, Chest 108:255 (1995), Coleman and Tsongalis, Molecular
Diagnostics (Human Press, Inc. 1996), Elles (ed.) Molecular
Diagnosis of Genetic Diseases (Humana Press, Inc. 1996), Landegren
(ed.), Laboratory Protocols for Mutation Detection (Oxford
University Press 1996), Birren et al. (eds.), Genome Analysis, Vol.
2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998),
Dracopoli et al. (eds.), Current Protocols in Human Genetics (John
Wiley & Sons 1998), and Richards and Ward, "Molecular
Diagnostic Testing," in Principles of Molecular Medicine, pages
83-88 (Humana Press, Inc. 1998)).
[0081] The protein truncation test is also useful for detecting the
inactivation of a gene in which translation-terminating mutations
produce only portions of the encoded protein (see, for example,
Stoppa-Lyonnet et al., Blood 91:3920 (1998)). According to this
approach, RNA is isolated from a biological sample, and used to
synthesize cDNA. PCR is then used to amplify the Zkun10 target
sequence and to introduce an RNA polymerase promoter, a translation
initiation sequence, and an in-frame ATG triplet. PCR products are
transcribed using an RNA polymerase, and the transcripts are
translated in vitro with a T7-coupled reticulocyte lysate system.
The translation products are then fractionated by SDS-PAGE to
determine the lengths of the translation products. The protein
truncation test is described, for example, by Dracopoli et al.
(eds.), Current Protocols in Human Genetics, pages 9.11.1 - 9.11.18
(John Wiley & Sons 1998).
[0082] The present invention also contemplates kits for performing
a diagnostic assay for Zkun10 gene expression or to analyze the
Zkun10 locus of a subject. Such kits comprise nucleic acid probes,
such as double-stranded nucleic acid molecules comprising the
nucleotide sequence of SEQ ID NOS:1 or 9, or a fragment thereof, as
well as single-stranded nucleic acid molecules having the
complement of the nucleotide sequence of SEQ ID NOS:1 or 9, or a
fragment thereof. Probe molecules may be DNA, RNA,
oligonucleotides, and the like. Kits may comprise nucleic acid
primers for performing PCR.
[0083] Such a kit can contain all the necessary elements to perform
a nucleic acid diagnostic assay described above. A kit will
comprise at least one container comprising a Zkun10 probe or
primer. The kit may also comprise a second container comprising one
or more reagents capable of indicating the presence of Zkun10
sequences. Examples of such indicator reagents include detectable
labels such as radioactive labels, fluorochromes, chemiluminescent
agents, and the like. A kit may also comprise a means for conveying
to the user that the Zkun10 probes and primers are used to detect
Zkun10 gene expression. For example, written instructions may state
that the enclosed nucleic acid molecules can be used to detect
either a nucleic acid molecule that encodes Zkun10, or a nucleic
acid molecule having a nucleotide sequence that is complementary to
a Zkun10-encoding nucleotide sequence, or to analyze chromosomal
sequences associated with the Zkun10 locus. The written material
can be applied directly to a container, or the written material can
be provided in the form of a packaging insert.
[0084] Zkun10 proteins, including variants of wild-type zkun10, are
tested for activity in protease inhibition assays, a variety of
which are known in the art. Preferred assays include those
measuring inhibition of trypsin, chymotrypsin, plasmin, cathepsin
G, and human leukocyte elastase. See, for example, Petersen et al.,
Eur. J. Biochem. 235:310-316, 1996. In a typical procedure, the
inhibitory activity of a test compound is measured by incubating
the test compound with the proteinase, then adding an appropriate
substrate, typically a chromogenic peptide substrate. See, for
example, Norris et al. (Biol. Chem. Hoppe-Seyler 371:37-42, 1990).
Briefly, various concentrations of the inhibitor are incubated in
the presence of trypsin, plasmin, and plasma kallikrein in a
low-salt buffer at pH 7.4, 25.degree. C. After 30 minutes, the
residual enzymatic activity is measured by the addition of a
chromogenic substrate (e.g., S2251 (D-Val-Leu-Lys-Nan) or S2302
(D-Pro-Phe-Arg-Nan), available from Kabi, Stockholm, Sweden) and a
30-minute incubation. Inhibition of enzyme activity is indicated by
a decrease in absorbance at 405 nm or fluorescence Em at 460 nm.
From the results, the apparent inhibition constant K.sub.i is
calculated. The inhibition of coagulation factors (e.g., factor
VIIa, factor Xa) can be measured using chromogenic substrates or in
conventional coagulation assays (e.g., clotting time of normal
human plasma; Dennis et al., ibid.).
[0085] Zknu10 proteins can be tested in animal models of disease,
particularly tumor models, models of fibrinolysis, and models of
imbalance of hemostasis. Suitable models are known in the art. For
example, inhibition of tumor metastasis can be assessed in mice
into which cancerous cells or tumor tissue have been introduced by
implantation or injection (e.g., Brown, Advan. Enzyme Regul.
35:293-301, 1995; Conway et al., Clin. Exp. Metastasis 14:115-124,
1996). Effects on fibrinolysis can be measured in a rat model
wherein the enzyme batroxobin and radiolabeled fibrinogen are
administered to test animals. Inhibition of fibrinogen activation
by a test compound is seen as a reduction in the circulating level
of the label as compared to animals not receiving the test
compound. See, Lenfors and Gustafsson, Semin. Thromb. Hemost.
22:335-342, 1996. Zkun10 proteins can be delivered to test animals
by injection or infusion, or can be produced in vivo by way of, for
example, viral or naked DNA delivery systems or transgenic
expression.
[0086] Exemplary viral delivery systems include adenovirus,
herpesvirus, vaccinia virus and adeno-associated virus (AAV).
Adenovirus, a double-stranded DNA virus, is currently the best
studied gene transfer vector for delivery of heterologous nucleic
acid (for a review, see Becker et al., Meth. Cell Biol. 43:161-189,
1994; and Douglas and Curiel, Science & Medicine 4:44-53,
1997). The adenovirus system offers several advantages: adenovirus
can (i) accommodate relatively large DNA inserts; (ii) be grown to
high titer; (iii) infect a broad range of mammalian cell types; and
(iv) be used with a large number of available vectors containing
different promoters. Also, because adenoviruses are stable in the
bloodstream, they can be administered by intravenous injection. By
deleting portions of the adenovirus genome, larger inserts (up to 7
kb) of heterologous DNA can be accommodated. These inserts can be
incorporated into the viral DNA by direct ligation or by homologous
recombination with a co-transfected plasmid. In an exemplary
system, the essential E1 gene is deleted from the viral vector, and
the virus will not replicate unless the E1 gene is provided by the
host cell (e.g., the human 293 cell line). When intravenously
administered to intact animals, adenovirus primarily targets the
liver. If the adenoviral delivery system has an E1 gene deletion,
the virus cannot replicate in the host cells. However, the host's
tissue (e.g., liver) will express and process (and, if a signal
sequence is present, secrete) the heterologous protein. Secreted
proteins will enter the circulation in the highly vascularized
liver, and effects on the infected animal can be determined.
[0087] An alternative method of gene delivery comprises removing
cells from the body and introducing a vector into the cells as a
naked DNA plasmid. The transformed cells are then re-implanted in
the body. Naked DNA vectors are introduced into host cells by
methods known in the art, including transfection, electroporation,
microinjection, transduction, cell fusion, DEAE dextran, calcium
phosphate precipitation, use of a gene gun, or use of a DNA vector
transporter. See, Wu et al., J. Biol. Chem. 263:14621-14624, 1988;
Wu et al., J. Biol. Chem. 267:963-967, 1992; and Johnston and Tang,
Meth. Cell Biol. 43:353-365, 1994.
[0088] Transgenic mice, engineered to express a zkun10 gene, and
mice that exhibit a complete absence of zkun10 gene function,
referred to as "knockout mice" (Snouwaert et al., Science 257:1083,
1992), can also be generated (Lowell et al., Nature 366:740-742,
1993). These mice are employed to study the zkun10 gene and the
encoded protein in an in vivo system. Transgenic mice are
particularly useful for investigating the role of zkun10 proteins
in early development because they allow the identification of
developmental abnormalities or blocks resulting from the over- or
underexpression of a specific factor.
[0089] The zkun10 polypeptides of the present invention, including
full-length polypeptides, biologically active fragments, and fusion
polypeptides can be produced in genetically engineered host cells
according to conventional techniques. Suitable host cells are those
cell types that can be transformed or transfected with exogenous
DNA and grown in culture, and include bacteria, fungal cells, and
cultured higher eukaryotic cells. Eukaryotic cells, particularly
cultured cells of multicellular organisms, are preferred.
Techniques for manipulating cloned DNA molecules and introducing
exogenous DNA into a variety of host cells are disclosed by
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989, and Ausubel et al., eds., Current Protocols in Molecular
Biology, John Wiley and Sons, Inc., NY, 1987.
[0090] In general, a DNA sequence encoding a zkun10 polypeptide is
operably linked to other genetic elements required for its
expression, generally including a transcription promoter and
terminator, within an expression vector. The vector will also
commonly contain one or more selectable markers and one or more
origins of replication, although those skilled in the art will
recognize that within certain systems selectable markers may be
provided on separate vectors, and replication of the exogenous DNA
may be provided by integration into the host cell genome. Selection
of promoters, terminators, selectable markers, vectors and other
elements is a matter of routine design within the level of ordinary
skill in the art. Many such elements are described in the
literature and are available through commercial suppliers.
[0091] To direct a zkun10 polypeptide into the secretory pathway of
a host cell, a secretory signal sequence (also known as a leader
sequence, prepro sequence or pre sequence) is provided in the
expression vector. The secretory signal sequence may be that of
zkun10, or may be derived from another secreted protein (e.g.,
t-PA) or synthesized de novo. The secretory signal sequence is
operably linked to the zkun10 DNA sequence, i.e., the two sequences
are joined in the correct reading frame and positioned to direct
the newly sythesized polypeptide into the secretory pathway of the
host cell. Secretory signal sequences are commonly positioned 5' to
the DNA sequence encoding the polypeptide of interest, although
certain signal sequences may be positioned elsewhere in the DNA
sequence of interest (see, e.g., Welch et al., U.S. Pat. No.
5,037,743; Holland et al., U.S. Pat. No. 5,143,830).
[0092] Cultured mammalian cells are suitable hosts for use within
the present invention. Methods for introducing exogenous DNA into
mammalian host cells include calcium phosphate-mediated
transfection (Wigler et al., Cell 14:725, 1978; Corsaro and
Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb,
Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et
al., ibid.), and liposome-mediated transfection (Hawley-Nelson et
al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993). The
production of recombinant polypeptides in cultured mammalian cells
is disclosed, for example, by Levinson et al., U.S. Pat. No.
4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al.,
U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134.
Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL
1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570
(ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J.
Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1;
ATCC No. CCL 61) cell lines. Additional suitable cell lines are
known in the art and available from public depositories such as the
American Type Culture Collection, 10801 University Boulevard,
Manassas, Va. In general, strong transcription promoters are
preferred, such as promoters from SV-40 or cytomegalovirus. See,
e.g., U.S. Pat. No. 4,956,288. Other suitable promoters include
those from metallothionein genes (U.S. Pat. Nos. 4,579,821 and
4,601,978) and the adenovirus major late promoter. Expression
vectors for use in mammalian cells include pZP-1 and pZP-9, which
have been deposited with the American Type Culture Collection,
10801 University Boulevard, Manassas, Va. under accession numbers
98669 and 98668, respectively.
[0093] Drug selection is generally used to select for cultured
mammalian cells into which foreign DNA has been inserted. Such
cells are commonly referred to as "transfectants". Cells that have
been cultured in the presence of the selective agent and are able
to pass the gene of interest to their progeny are referred to as
"stable transfectants." A preferred selectable marker is a gene
encoding resistance to the antibiotic neomycin. Selection is
carried out in the presence of a neomycin-type drug, such as G-418
or the like. Selection systems can also be used to increase the
expression level of the gene of interest, a process referred to as
"amplification." Amplification is carried out by culturing
transfectants in the presence of a low level of the selective agent
and then increasing the amount of selective agent to select for
cells that produce high levels of the products of the introduced
genes. A preferred amplifiable selectable marker is dihydrofolate
reductase, which confers resistance to methotrexate. Other drug
resistance genes (e.g. hygromycin resistance, multi-drug
resistance, puromycin acetyltransferase) can also be used.
[0094] Other higher eukaryotic cells can also be used as hosts,
including insect cells, plant cells and avian cells. The use of
Agrobacterium rhizogenes as a vector for expressing genes in plant
cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore)
11:47-58, 1987. Insect cells can be infected with recombinant
baculovirus vectors, which are commonly derived from Autographa
californica multiple nuclear polyhedrosis virus (AcMNPV). DNA
encoding the polypeptide of interest is inserted into the viral
genome in place of the polyhedrin gene coding sequence by
homologous recombination in cells infected with intact, wild-type
AcMNPV and transfected with a transfer vector comprising the cloned
gene operably linked to polyhedrin gene promoter, terminator, and
flanking sequences. The resulting recombinant virus is used to
infect host cells, typically a cell line derived from the fall
armyworm, Spodoptera frugiperda. See, in general, Glick and
Pasternak, Molecular Biotechnology: Principles and Applications of
Recombinant DNA, ASM Press, Washington, D.C., 1994.
[0095] Fungal cells, including yeast cells, can also be used within
the present invention. Yeast species of particular interest in this
regard include Saccharomyces cerevisiae, Pichia pastoris, and
Pichia methanolica. Methods for transforming S. cerevisiae cells
with exogenous DNA and producing recombinant polypeptides therefrom
are disclosed by, for example, Kawasaki, U.S. Pat. No. 4,599,311;
Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No.
4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et
al., U.S. Pat. No. 4,845,075. Transformed cells are selected by
phenotype determined by the selectable marker, commonly drug
resistance or the ability to grow in the absence of a particular
nutrient (e.g., leucine). A preferred vector system for use in
Saccharomyces cerevisiae is the POT1 vector system disclosed by
Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformed
cells to be selected by growth in glucose-containing media.
Suitable promoters and terminators for use in yeast include those
from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No.
4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; and Bitter,
U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes. See also
U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.
Transformation systems for other yeasts, including Hansenula
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia
methanolica, Pichia guillernondii and Candida maltosa are known in
the art. See, for example, Gleeson et al., J. Gen. Microbiol.
132:3459-3465, 1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus
cells may be utilized according to the methods of McKnight et al.,
U.S. Pat. No. 4,935,349. Methods for transforming Acremonium
chrysogenum are disclosed by Sumino et al., U.S. Pat. No.
5,162,228. Methods for transforming Neurospora are disclosed by
Lambowitz, U.S. Pat. No. 4,486,533. Production of recombinant
proteins in Pichia methanolica is disclosed in U.S. Pat. Nos.
5,716,808, 5,736,383, 5,854,039, and 5,888,768.
[0096] Prokaryotic host cells, including strains of the bacteria
Escherichia coli, Bacillus and other genera are also useful host
cells within the present invention. Techniques for transforming
these hosts and expressing foreign DNA sequences cloned therein are
well known in the art (see, e.g., Sambrook et al., ibid.). When
expressing a zkun10 polypeptide in bacteria such as E. coli, the
polypeptide may be retained in the cytoplasm, typically as
insoluble granules, or may be directed to the periplasmic space by
a bacterial secretion sequence. In the former case, the cells are
lysed, and the granules are recovered and denatured using, for
example, guanidine isothiocyanate or urea. The denatured
polypeptide can then be refolded and dimerized by diluting the
denaturant, such as by dialysis against a solution of urea and a
combination of reduced and oxidized glutathione, followed by
dialysis against a buffered saline solution. In the latter case,
the polypeptide can be recovered from the periplasmic space in a
soluble and functional form by disrupting the cells (by, for
example, sonication or osmotic shock) to release the contents of
the periplasmic space and recovering the protein, thereby obviating
the need for denaturation and refolding.
[0097] Transformed or transfected host cells are cultured according
to conventional procedures in a culture medium containing nutrients
and other components required for the growth of the chosen host
cells. A variety of suitable media, including defined media and
complex media, are known in the art and generally include a carbon
source, a nitrogen source, essential amino acids, vitamins and
minerals. Media may also contain such components as growth factors
or serum, as required. The growth medium will generally select for
cells containing the exogenously added DNA by, for example, drug
selection or deficiency in an essential nutrient which is
complemented by the selectable marker carried on the expression
vector or co-transfected into the host cell. P. methanolica cells
are cultured in a medium comprising adequate sources of carbon,
nitrogen and trace nutrients at a temperature of about 25.degree.
C. to 35.degree. C. Liquid cultures are provided with sufficient
aeration by conventional means, such as shaking of small flasks or
sparging of fermentors.
[0098] It is preferred to purify the proteins of the present
invention to .gtoreq.80% purity, more preferably to .gtoreq.90%
purity, even more preferably .gtoreq.95% purity, and particularly
preferred is a pharmaceutically pure state, that is greater than
99.9% pure with respect to contaminating macromolecules,
particularly other proteins and nucleic acids, and free of
infectious and pyrogenic agents. Preferably, a purified protein is
substantially free of other proteins, particularly other proteins
of animal origin.
[0099] Zknu10 proteins are purified by conventional protein
purification methods, typically by a combination of chromatographic
techniques. Polypeptides comprising a polyhistidine affinity tag
(typically about 6 histidine residues) are purified by affinity
chromatography on a nickel chelate resin. See, for example,
Houchuli et al., Bio/Technol. 6: 1321-1325, 1988.
[0100] Using methods known in the art, zkun10 proteins can be
produced glycosylated or non-glycosylated; pegylated or
non-pegylated; and may or may not include an initial methionine
amino acid residue.
[0101] The zkun10 proteins are contemplated for use in the
treatment or prevention of conditions associated with excessive
proteinase activity, in particular an excess of trypsin, plasmin,
kallikrein, elastase, cathepsin G, proteinase-3, thrombin, factor
VIIa, factor IXa, factor Xa, factor XIa, factor XIIa, or matrix
metalloproteinases. Such conditions include, but are not limited
to, acute pancreatitis, cardiopulmonary bypass (CPB)-induced
pulmonary injury, allergy-induced protease release, deep vein
thrombosis, myocardial infarction, shock (including septic shock),
hyperfibrinolytic hemorrhage, emphysema, rheumatoid arthritis,
adult respiratory distress syndrome, chronic inflammatory bowel
disease, psoriasis, and other inflammatory conditions. Zknu10
proteins are also contemplated for use in preservation of platelet
function, organ preservation, and wound healing.
[0102] Zkun10 proteins may be useful in the treatment of conditions
arising from an imbalance in hemostasis, including acquired
coagulopathies, primary fibrinolysis and fibrinolysis due to
cirrhosis, and complications from high-dose thrombolytic therapy.
Acquired coagulopathies can result from liver disease, uremia,
acute disseminated intravascular coagulation, post-cardiopulmonary
bypass, massive transfusion, or Warfarin overdose (Humphries,
Transfusion Medicine 1:1181-1201, 1994). A deficiency or
dysfunction in any of the procoagulant mechanisms predisposes the
patient to either spontaneous hemorrhage or excess blood loss
associated with trauma or surgery. Acquired coagulopathies usually
involve a combination of deficiencies, such as deficiencies of a
plurality of coagulation factors, and/or platelet dysfunction. In
addition, patients with liver disease commonly experience increased
fibrinolysis due to an inability to maintain normal levels of
a.sub.2-antiplasmin and/or decreased hepatic clearance of
plasminogen activators (Shuman, Hemorrhagic Disorders, in Bennet
and Plum, eds. Cecil Textbook of Medicine, 20th ed., W. B. Saunders
Co., 1996). Primary fibrinolysis results from a massive release of
plasminogen activator. Conditions associated with primary
fibrinolysis include carcinoma of the prostate, acute promyelocytic
leukemia, hemangiomas, and sustained release of plasminogen
activator by endothelial cells due to injection of venoms. The
condition becomes critical when enough plasmin is activated to
deplete the circulating level of .alpha..sub.2-antiplasmi- n
(Shuman, ibid.). Data suggest that plasmin on endothelial cells may
be related to the pathophysiology of bleeding or rethrombosis
observed in patients undergoing high-dose thrombolytic therapy for
thrombosis. Plasmin may cause further damage to the thrombogenic
surface of blood vessels after thrombolysis, which may result in
rethrombosis (Okajima, J. Lab. Clin. Med. 126:1377-1384, 1995).
[0103] Additional antithrombotic uses of zkun10 proteins include
treatment or prevention of deep vein thrombosis, pulmonary
embolism, post-surgical thrombosis, and regulation of blood
pressure.
[0104] Zkun10 proteins may also be used within methods for
inhibiting blood coagulation in mammals, such as in the treatment
of disseminated intravascular coagulation. Zkun10 proteins may thus
be used in place of known anticoagulants such as heparin, coumarin,
and anti-thrombin III. Such methods will generally include
administration of the protein in an amount sufficient to produce a
clinically significant inhibition of blood coagulation. Such
amounts will vary with the nature of the condition to be treated,
but can be predicted on the basis of known assays and experimental
animal models, and will in general be within the ranges disclosed
below.
[0105] Zkun10 proteins may also find therapeutic use in the
blockage of proteolytic tissue degradation. Proteolysis of
extracellular matrix, connective tissue, and other tissues and
organs is an element of many diseases. This tissue destruction is
beleived to be initiated when plasmin activates one or more matrix
metalloproteinases (e.g., collagenase and metallo-elastases).
Inhibition of plasmin by zkun10 proteins may thus be beneficial in
the treatment of these conditions.
[0106] Matrix metalloproteinases (MMPs) are believed to play a role
in metastases of cancers, abdominal aortic aneurysm, multiple
sclerosis, rheumatoid arthritis, osteoarthritis, trauma and
hemorrhagic shock, and corneal ulcers. MMPs produced by tumor cells
break down and remodel tissue matrices during the process of
metastatic spread. There is evidence to suggest that MMP inhibitors
may block this activity (Brown, Advan. Enzyme Regul. 35:293-301,
1995). Abdominal aortic aneurysm is characterized by the
degradation of extracellular matrix and loss of structural
integrity of the aortic wall. Data suggest that plasmin may be
important in the sequence of events leading to this destruction of
aortic matrix (Jean-Claude et al., Surgery 116:472-478, 1994).
Proteolytic enzymes are also believed to contribute to the
inflammatory tissue damage of multiple sclerosis (Gijbels, J. Clin.
Invest. 94:2177-2182, 1994). Rheumatoid arthritis is a chronic,
systemic inflammatory disease predominantly affecting joints and
other connective tissues, wherein proliferating inflammatory tissue
(panus) may cause joint deformities and dysfunction (see, Arnett,
in Cecil Textbook of Medicine, ibid.). Osteoarthritis is a chronic
disease causing deterioration of the joint cartilage and other
joint tissues and the formation of new bone (bone spurs) at the
margins of the joints. There is evidence that MMPs participate in
the degradation of collagen in the matrix of osteoarthritic
articular cartilage. Inhition of MMPs results in the inhibition of
the removal of collagen from cartilage matrix (Spirito, Inflam.
Res. 44 (supp. 2):S131-S132, 1995; O'Byrne, Inflam. Res. 44 (supp.
2):S117-S118, 1995; Karran, Ann. Rheumatic Disease 54:662-669,
1995). Zkun10 proteins may also be useful in the treatment of
trauma and hemorrhagic shock. Data suggest that administration of
an MMP inhibitor after hemorrhage improves cardiovascular response,
hepatocellular function, and microvascular blood flow in various
organs (Wang, Shock 6:377-382, 1996). Corneal ulcers, which can
result in blindness, manifest as a breakdown of the collagenous
stromal tissue. Damage due to thermal or chemical injury to corneal
surfaces often results in a chronic wound-healing situation. There
is direct evidence for the role of MMPs in basement membrane
defects associated with failure to re-epithelialize in cornea or
skin (Fini, Am. J. Pathol. 149:1287-1302, 1996).
[0107] The zkun10 proteins of the present invention may be combined
with other therapeutic agents to augment the activity (e.g.,
antithrombotic or anticoagulant activity) of such agents. For
example, a zkun10 protein may be used in combination with tissue
plasminogen activator in thrombolytic therapy.
[0108] Doses of zkun10 proteins will vary according to the severity
of the condition being treated and may range from approximately 10
.mu.g/kg to 10 mg/kg body weight, preferably 100 .mu.g/kg to 5
mg/kg, more preferably 100 .mu.g/kg to 1 mg/kg. The proteins
formulated in a pharmaceutically acceptable carrier or vehicle. It
is preferred to prepare them in a form suitable for injection or
infusion, such as by dilution with with sterile water, an isotonic
saline or glucose solution, or similar vehicle. In the alternative,
the protein may be packaged as a lyophilized powder, optionally in
combination with a pre-measured diluent, and resuspended
immediately prior to use. Pharmaceutical compositions may further
include one or more excipients, preservatives, solubilizers,
buffering agents, albumin to prevent protein loss on vial surfaces,
etc. Formulation methods are within the level of ordinary skill in
the art. See, Remington: The Science and Practice of Pharmacy,
Gennaro, ed., Mack Publishing Co., Easton, Pa., 19th ed., 1995.
[0109] Gene therapy provides an alternative therapeutic approach
for delivery of zkun10 proteins. If a mammal has a mutated or
absent zkun10 gene, a polynucleotide encoding a zkun10 protein can
be introduced into the cells of the mammal. In one embodiment, a
gene encoding a zkun10 protein is introduced in vivo in a viral
vector. Such vectors include an attenuated or defective DNA virus,
such as herpes simplex virus (HSV), papillomavirus, Epstein Barr
virus (EBV), adenovirus, adeno-associated virus (AAV), and the
like. Defective viruses, which entirely or almost entirely lack
viral genes, are preferred. A defective virus is not infective
after introduction into a cell. Use of defective viral vectors
allows for administration to cells in a specific, localized area,
without concern that the vector can infect other cells. Examples of
particular vectors include, without limitation, a defective herpes
simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell.
Neurosci. 2:320-30, 1991); an attenuated adenovirus vector, such as
the vector described by Stratford-Perricaudet et al., J. Clin.
Invest. 90:626-30, 1992; and a defective adeno-associated virus
vector (Samulski et al., J. Virol. 61:3096-101, 1987; Samulski et
al., J. Virol. 63:3822-8, 1989).
[0110] Within another embodiment, a zkun10 polynucleotide can be
introduced in a retroviral vector, as described, for example, by
Anderson et al., U.S. Pat. No. 5,399,346; Mann et al. Cell 33:153,
1983; Temin et al., U.S. Pat. No. 4,650,764; Temin et al., U.S.
Pat. No. 4,980,289; Markowitz et al., J. Virol. 62:1120, 1988;
Temin et al., U.S. Pat. No. 5,124,263; Dougherty et al., WIPO
Publication No. WO 95/07358; and Kuo et al., Blood 82:845, 1993.
Alternatively, the vector can be introduced by lipofection in vivo
using liposomes. Synthetic cationic lipids can be used to prepare
liposomes for in vivo transfection of a gene encoding a marker
(Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey
et al., Proc. Natl. Acad. Sci. USA 85:8027-31, 1988).
[0111] Within a further embodiment, target cells are removed from
the body, and a vector is introduced into the cells as a naked DNA
plasmid. The transformed cells are then re-implanted into the body.
Naked DNA vectors for gene therapy can be introduced into the
desired host cells by methods known in the art, e.g., transfection,
electroporation, microinjection, transduction, cell fusion, DEAE
dextran, calcium phosphate precipitation, use of a gene gun or use
of a DNA vector transporter. See, for example, Wu et al., J. Biol.
Chem. 267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4,
1988.
[0112] Zkun10 proteins can also be used to prepare antibodies that
specifically bind to zkun10 proteins. As used herein, the term
"antibodies" includes polyclonal antibodies, monoclonal antibodies,
antigen-binding fragments thereof such as F(ab').sub.2 and Fab
fragments, single chain antibodies, and the like, including
genetically engineered antibodies. Non-human antibodies can be
humanized by grafting only non-human CDRs onto human framework and
constant regions, or by incorporating the entire non-human variable
domains (optionally "cloaking" them with a human-like surface by
replacement of exposed residues, wherein the result is a "veneered"
antibody). In some instances, humanized antibodies may retain
non-human residues within the human variable region framework
domains to enhance proper binding characteristics. Through
humanizing antibodies, biological half-life may be increased, and
the potential for adverse immune reactions upon administration to
humans is reduced. One skilled in the art can generate humanized
antibodies with specific and different constant domains (i.e.,
different Ig subclasses) to facilitate or inhibit various immune
functions associated with particular antibody constant domains.
Alternative techniques for generating or selecting antibodies
useful herein include in vitro exposure of lymphocytes to a zkun10
protein, and selection of antibody display libraries in phage or
similar vectors (for instance, through use of immobilized or
labeled zkun10 polypeptide). Antibodies are defined to be
specifically binding if they bind to a zkun10 protein with an
affinity at least 10-fold greater than the binding affinity to
control (non-zkun10) polypeptide. It is preferred that the
antibodies exhibit a binding affinity (K.sub.a) of 10.sup.6
M.sup.-1 or greater, preferably 10.sup.7 M.sup.-1 or greater, more
preferably 10.sup.8 M.sup.- or greater, and most preferably
10.sup.9 M.sup.-1 or greater. The affinity of a monoclonal antibody
can be readily determined by one of ordinary skill in the art (see,
for example, Scatchard, Ann. NY Acad. Sci. 51: 660-672,1949).
[0113] Methods for preparing polyclonal and monoclonal antibodies
are well known in the art (see for example, Hurrell, J. G. R., Ed.,
Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC
Press, Inc., Boca Raton, Fla., 1982). As would be evident to one of
ordinary skill in the art, polyclonal antibodies can be generated
from a variety of warm-blooded animals such as horses, cows, goats,
sheep, dogs, chickens, rabbits, mice, and rats. The immunogenicity
of a zkun10 protein may be increased through the use of an adjuvant
such as alum (aluminum hydroxide) or Freund's complete or
incomplete adjuvant. Polypeptides useful for immunization also
include fusion polypeptides, such as fusions of a zkun10 protein or
a portion thereof with an immunoglobulin polypeptide or with
maltose binding protein. The polypeptide immunogen may be a
full-length molecule or a portion thereof. If the polypeptide
portion is "hapten-like", such portion may be advantageously joined
or linked to a macromolecular carrier (such as keyhole limpet
hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for
immunization.
[0114] Immunogenic zkun10 polypeptides may be as small as 5
residues. It is preferred to use polypeptides that are hydrophilic
or comprise a hydrophilic region. A preferred such region of SEQ ID
NO:2 includes residues 44 (Asn)-54 (Asp).
[0115] A variety of assays known to those skilled in the art can be
utilized to detect antibodies that specifically bind to a zkun10
protein. Exemplary assays are described in detail in Antibodies: A
Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor
Laboratory Press, 1988. Representative examples of such assays
include concurrent immunoelectrophoresis, radio-immunoassays,
radio-immunoprecipitations, enzyme-linked immunosorbent assays
(ELISA), dot blot assays, Western blot assays, inhibition or
competition assays, and sandwich assays.
[0116] Antibodies to zkun10 may be used for affinity purification
of zkun10 proteins; within diagnostic assays for determining
circulating levels of zkun10 proteins; for detecting or
quantitating soluble zkun10 protein as a marker of underlying
pathology or disease; for immunolocalization within whole animals
or tissue sections, including immunodiagnostic applications; for
immunohistochemistry; for screening expression libraries; and for
other uses that will be evident to those skilled in the art. For
certain applications, including in vitro and in vivo diagnostic
uses, it is advantageous to employ labeled antibodies. Suitable
direct tags or labels include radionuclides, enzymes, substrates,
cofactors, inhibitors, fluorescent markers, chemiluminescent
markers, magnetic particles and the like; indirect tags or labels
may feature use of biotin-avidin or other
complement/anti-complement pairs as intermediates.
[0117] Zkun10 proteins may be used in the laboratory or commercial
preparation of proteins from cultured cells. The proteins can be
used alone to inhibit specific proteolysis or can be combined with
other proteinase inhibitors to provide a "cocktail" with a broad
spectrum of activity. Of particular interest is the inhibition of
cellular proteases, which can be release during cell lysis. The
proteins can also be used in the laboratory as a tissue culture
additive to prevent cell detachment.
[0118] Zkun10 polypeptides can also be used to teach analytical
skills such as mass spectrometry, circular dichroism, to determine
conformation, especially of the four alpha helices, x-ray
crystallography to determine the three-dimensional structure in
atomic detail, nuclear magnetic resonance spectroscopy to reveal
the structure of proteins in solution. For example, a kit
containing the Zkun10 can be given to the student to analyze. Since
the amino acid sequence would be known by the instructor, the
protein can be given to the student as a test to determine the
skills or develop the skills of the student, the instructor would
then know whether or not the student has correctly analyzed the
polypeptide. Since every polypeptide is unique, the educational
utility of Zkun10 would be unique unto itself.
[0119] The antibodies which bind specifically to Zkun10 can be used
as a teaching aid to instruct students how to prepare affinity
chromatography columns to purify Zkun10, cloning and sequencing the
polynucleotide that encodes an antibody and thus as a practicum for
teaching a student how to design humanized antibodies. The Zkun10
gene, polypeptide, or antibody would then be packaged by reagent
companies and sold to educational institutions so that the students
gain skill in art of molecular biology. Because each gene and
protein is unique, each gene and protein creates unique challenges
and learning experiences for students in a lab practicum. Such
educational kits containing the Zkun10 gene, polypeptide, or
antibody are considered within the scope of the present
invention.
[0120] In summary, the present invention provides isolated
polypeptides comprising a sequence of amino acid residues as shown
in SEQ ID NO: 2 from residue 57 to residue 107. In other
embodiments, the present invention includes polypeptides that have
at least 95% identity to SEQ ID NO: 2 from residue 1 to residue
111. Changes with the amino acid sequence of SEQ ID NO: 2 from
residue 57 to residue 107, or polynucleotides encoding that
sequence, will be subject to the limitations as shown in SEQ ID NO:
4, which is illustrative of a motif for the Kunitz domain. Other
embodiments include isolated polypeptides with 95% identity to
those shown in SEQ ID NO: 6 from residues 1 to 415, which include a
Kunitz domain as shown in residues 361 to 415 (and are subject to
the same limitations as described herein for SEQ ID NO: 2 residues
57 to 107).
[0121] In another aspect, the present invention includes fusion
proteins comprising at least polypeptides, of which at least one
polypeptide comprises a sequence of amino acid residues as shown in
SEQ ID NO: 2 from residue 57 to residue 107. Other embodiment
include fusion proteins of at least three polypeptides, wherein the
first polypeptide comprises a secretory signal sequence; the second
polypeptide comprises a collagen domain containing one or more von
Willebrand domains; and a third polypeptide comprising one or more
Kunitz domains, one of which comprises the sequence of amino acid
residues as shown in SEQ ID NO: 2 from residue 57 to 107. In
additional embodiments, the fusion protein will contain collagen
globular domains.
[0122] In another aspect, the present invention provides expression
vectors comprising a transcription promoter, a DNA segment encoding
for polypeptides as described above, and a transcription
terminator. In another aspect, the expression vector will expressed
in a cultured cell. In another aspect, the present invention
includes methods by which the polypeptide expressed by the cultured
is recovered. The present invention also provides antibodies which
specifically bind the polypeptides described herein.
[0123] In other aspects, the present invention provides
polynucleotide molecules that encode for the polypeptides described
herein. In certain embodiments the polynucleotides comprise a
sequence of nucleotides as shown in SEQ ID NO: 1 from nucleotide
169 to nucleotide 321 or as shown in SEQ ID NO: 1 from nucleotide 1
to nucleotide 333; or as shown in SEQ ID NO: 5 from nucleotide 1 to
nucleotide 1248.
[0124] The present invention also provides methods for inhibiting
protease degradation, in particular in compositions that contain
plasma proteins. These compositions will have a composition
comprising zkun10 polypeptides as described herein added to the
protein composition in an amount sufficient to reduce the
degradation of the protein composition by proteases. The reduction
in protease degradation or activity can be measured using
chromogenic assays or clotting assays.
[0125] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
Cloning Zkun10
[0126] To obtain a Zkun10 cDNA clone, cDNA is prepared from stomach
using a a commercially available kit (Marathon.TM. cDNA
Amplification Kit from Clontech Laboratories, Inc., Palo Alto,
Calif.) and an oligo(dT) primer. To amplify the zkun10 DNA, 5 .mu.l
each of {fraction (1/100)} diluted cDNAs, 20 pmoles each of two
oligonucleotide primers designed from SEQ ID NO:1, and 1 U of a 2:1
mixture of ExTaq.TM. DNA polymerase (TaKaRa Biomedicals) and Pfu
DNA polymerasse (Stratagene, La Jolla, Calif.) (ExTaq/Pfu) are used
in a 25- .mu.l reaction mixture. The reaction mixture is incubated
at 94.degree. C. for 2 minutes; 25 cycles of 94.degree. C. for 15
seconds, 66.degree. C. for 20 seconds, and 72.degree. C. for 30
seconds; and a 1-minute incubation at 72.degree. C. 1 .mu.l each of
{fraction (1/100)} diluted first PCR product is used as template
for a nested PCR. 20 pmoles each of two additional oligonucleotide
primers and 1 U of ExTaqlPfu are used in 25-.mu.l reaction
mixtures. The mixtures are incubated at 94.degree. C. for 2
minutes; 2 cycles of 94.degree. C. for 15 seconds, 66.degree. C.
for 20 seconds, 72.degree. C. for 30 seconds; 25 cycles of
94.degree. C. for 15 seconds, 64.degree. C. for 20 seconds,
72.degree. C. for 30 seconds; and a 1-minute incubation at
72.degree. C. The PCR products are gel purified and sequenced to
confirm their identity.
[0127] To construct an expression vector for the zkun10 Kunitz
domain, PCR is performed on cDNA prepared from stomach as disclosed
above. Primers are designed such that the PCR product will encode
an intact Kunitz domain with restriction sites Bam HI in the sense
primer and Xho I in the antisense primer to facilitate subcloning
into an expression vector. 5 .mu.l of {fraction (1/100)} diluted
cDNA, 20 pmoles of each oligonucleotide primer, and 1 U of
ExTaqlPfu are used in 25-.mu.l reaction mixtures. The mixtures are
incubated at 94.degree. C. for 2 minutes; 3 cycles of 94.degree. C.
for 30 seconds, 50.degree. C. for 30 seconds, 72.degree. C. for 30
seconds; 35 cycles of 94.degree. C. for 30 seconds, 68.degree. C.
for 30 seconds; and a 7-minute incubation at 72.degree. C. The PCR
product is gel purified and restriction digested with Bam HI and
Xho I overnight.
[0128] A mammalian expression vector is constructed with the
dihyrofolate reductase gene selectable marker under control of the
SV40 early promoter, SV40 polyadenylation site, a cloning site to
insert the gene of interest under control of the mouse
metallothionein 1 (MT-1) promoter and the hGH polyadenylation site.
The expression vector is designated pZP-9 and has been deposited at
the American Type Culture Collection, 10801 University Boulevard,
Manassas, Va. under accession no 98668. To facilitate protein
purification, the pZP9 vector is modified by addition of a tissue
plasminogen activator (t-PA) secretory signal sequence (see U.S.
Pat. No. 5,641,655) and a GluGlu tag sequence (SEQ ID NO:6) between
the MT-1 promoter and hGH terminator. The t-PA secretory signal
sequence replaces the native secretory signal sequence for DNAs
encoding polypeptides of interest that are inserted into this
vector, and expression results in an N-terminally tagged protein.
The N-terminally tagged vector was designated pZP9NEE. The vector
pZPNEE is digested with Bam HI and Xho I, and the zkun10 fragment
is inserted. The resulting construct is confirmed by
sequencing.
Example 2
Expression of Zkun10 in CHO Cells
[0129] CHO DG44 cells (Chasin et al., Som. Cell. Molec. Genet.
12:555-666, 1986) are plated in 10-cm tissue culture dishes and
allowed to grow to approximately 50% to 70% confluency overnight at
37.degree. C., 5% CO.sub.2, in Ham's F12/FBS media (Ham's F12
medium (Life Technologies), 5% fetal bovine serum (Hyclone, Logan,
Utah), 1% L-glutamine (JRH Biosciences, Lenexa, Kans.), 1% sodium
pyruvate (Life Technologies)). The cells are then transfected with
the plasmid zkun10 /pZMP6 by liposome-mediated transfection using a
3:1 (w/w) liposome formulation of the polycationic lipid
2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-
-dimethyl-1-propaniminium-trifluoroacetate and the neutral lipid
dioleoyl phosphatidylethanolamine in membrane-filetered water
(Lipofectamine.TM. Reagent, Life Technologies), in serum free (SF)
media formulation (Ham's F12, 10 mg/ml transferrin, 5 mg/ml
insulin, 2 mg/mil fetuin, 1% L-glutamine and 1% sodium pyruvate).
Zkun10/pZMP6 is diluted into 15-ml tubes to a total final volume of
640 .mu.l with SF media. 35 .mu.l of Lipofectamine.TM. is mixed
with 605 .mu.l of SF medium. The resulting mixture is added to the
DNA mixture and allowed to incubate approximately 30 minutes at
room temperature. Five ml of SF media is added to the
DNA:Lipofectamine.TM. mixture. The cells are rinsed once with 5 ml
of SF media, aspirated, and the DNA:Lipofectamine.TM. mixture is
added. The cells are incubated at 37.degree. C. for five hours,
then 6.4 ml of Ham's F12/10% FBS, 1% PSN media is added to each
plate. The plates are incubated at 37.degree. C. overnight, and the
DNA:Lipofectamine.TM. mixture is replaced with fresh 5% FBS/Ham's
media the next day. On day 3 post-transfection, the cells are split
into T-175 flasks in growth medium. On day 7 postransfection, the
cells are stained with FITC-anti-CD8 monoclonal antibody
(Pharmingen, San Diego, Calif.) followed by anti-FITC-conjugated
magnetic beads (Miltenyi Biotec). The CD8-positive cells are
separated using commercially available columns (mini-MACS columns;
Miltenyi Biotec) according to the manufacturer's directions and put
into DMEM/Ham's F12/5% FBS without nucleosides but with 50 nM
methotrexate (selection medium).
[0130] Cells are plated for subcloning at a density of 0.5, 1 and 5
cells per well in 96-well dishes in selection medium and allowed to
grow out for approximately two weeks. The wells are checked for
evaporation of medium and brought back to 200 .mu.l per well as
necessary during this process. When a large percentage of the
colonies in the plate are near confluency, 100 .mu.l of medium is
collected from each well for analysis by dot blot, and the cells
are fed with fresh selection medium. The supernatant is applied to
a nitrocellulose filter in a dot blot apparatus, and the filter is
treated at 100.degree. C. in a vacuum oven to denature the protein.
The filter is incubated in 625 mM Tris-glycine, pH 9.1, 5 mM
.beta.-mercaptoethanol, at 65.degree. C., 10 minutes, then in 2.5%
non-fat dry milk Western A Buffer (0.25% gelatin, 50 mM Tris-HCl pH
7.4, 150 mM NaCl, 5 mM EDTA, 0.05% Igepal CA-630) overnight at
4.degree. C. on a rotating shaker. The filter is incubated with the
antibody-HRP conjugate in 2.5% non-fat dry milk Western A buffer
for 1 hour at room temperature on a rotating shaker. The filter is
then washed three times at room temperature in PBS plus 0.01% Tween
20, 15 minutes per wash. The filter is developed with
chemiluminescence reagents (ECL.TM. direct labelling kit; Amersham
Corp., Arlington Heights, Ill.) according to the manufacturer's
directions and exposed to film (Hyperfilm ECL, Amersham Corp.) for
approximately 5 minutes. Positive clones are trypsinized from the
96-well dish and transferred to 6-well dishes in selection medium
for scaleup and analysis by Western blot.
Example 3
Expression of Zkun10 in BHK Cells
[0131] Full-length zkun10 protein is produced in BHK cells
transfected with pZMP6/zkun10 (Example 1). BHK 570 cells (ATCC
CRL-10314) are plated in 10-cm tissue culture dishes and allowed to
grow to approximately 50 to 70% confluence overnight at 37.degree.
C., 5% CO.sub.2, in DMEM/FBS media (DMEM, Gibco/BRL High Glucose;
Life Technologies), 5% fetal bovine serum (Hyclone, Logan, Utah), 1
mM L-glutamine (JRH Biosciences, Lenexa, Kans.), 1 mM sodium
pyruvate (Life Technologies). The cells are then transfected with
pZMP6/zkun10 by liposome-mediated transfection (using
Lipofectamine.TM.; Life Technologies), in serum free (SF) media
(DMEM supplemented with 10 mg/ml transferrin, 5 mg/ml insulin, 2
mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate). The plasmid
is diluted into 15-ml tubes to a total final volume of 640 .mu.l
with SF media. 35 .mu.l of the lipid mixture is mixed with 605
.mu.l of SF medium, and the resulting mixture is allowed to
incubate approximately 30 minutes at room temperature. Five
milliliters of SF media is then added to the DNA:lipid mixture. The
cells are rinsed once with 5 ml of SF media, aspirated, and the
DNA:lipid mixture is added. The cells are incubated at 37.degree.
C. for five hours, then 6.4 ml of DMEM/10% FBS, 1% PSN media is
added to each plate. The plates are incubated at 37.degree. C.
overnight, and the DNA:lipid mixture is replaced with fresh 5%
FBS/DMEM media the next day. On day 5 post-transfection, the cells
are split into T-162 flasks in selection medium (DMEM+5% FBS, 1%
L-Gln, 1% NaPyr, 1 .mu.M methotrexate). Approximately 10 days
post-transfection, two 150-mm culture dishes of
methotrexate-resistant colonies from each transfection are
trypsinized, and the cells are pooled and plated into a T-162 flask
and transferred to large-scale culture.
Example 4
Expression of Zkun10 in Adenovirus
[0132] For construction of adenovirus vectors, the protein coding
region of human zkun10 is amplified by PCR using primers that add
PmeI and AscI restriction sites at the 5' and 3' termini
respectively. Amplification is performed with a full-length zkun10
cDNA template in a PCR reaction as follows: one cycle at 95.degree.
C. for 5 minutes; followed by 15 cycles at 95.degree. C. for 1
min., 61.degree. C. for 1 min., and 72.degree. C. for 1.5 min.;
followed by 72.degree. C. for 7 min.; followed by a 4.degree. C.
soak. The PCR reaction product is loaded onto a 1.2%
low-melting-temperature agarose gel in TAE buffer (0.04 M
Tris-acetate, 0.001 M EDTA). The zkun10 PCR product is excised from
the gel and purified using a commercially available kit comprising
a silica gel mambrane spin column (QIAquick.RTM. PCR Purification
Kit and gel cleanup kit; Qiagen, Inc.) as per kit instructions. The
PCR product is then digested with PmeI and AscI, phenol/chloroform
extracted, EtOH precipitated, and rehydrated in 20 ml TE (Tris/EDTA
pH 8). The zkun10 fragment is then ligated into the PmeI-AscI sites
of the transgenic vector pTG12-8 and transformed into E. coli
DH10B.TM. competent cells by electroporation. Vector pTG12-8 was
derived from p2999B4 (Palmiter et al., Mol. Cell Biol.
13:5266-5275, 1993) by insertion of a rat insulin II intron (ca.
200 bp) and polylinker (Fse I/Pme I/Asc I) into the Nru I site. The
vector comprises a mouse metallothionein (MT-1) promoter (ca. 750
bp) and human growth hormone (hGH) untranslated region and
polyadenylation signal (ca. 650 bp) flanked by 10 kb of MT-1 5'
flanking sequence and 7 kb of MT-1 3' flanking sequence. The cDNA
is inserted between the insulin II and hGH sequences. Clones
containing zkun10 are identified by plasmid DNA miniprep followed
by digestion with PmeI and AscI. A positive clone is sequenced to
insure that there were no deletions or other anomalies in the
construct.
[0133] DNA is prepared using a commercially available kit (Maxi
Kit, Qiagen, Inc.), and the zkun10 cDNA is released from the
pTG12-8 vector using PmeI and AscI enzymes. The cDNA is isolated on
a 1% low melting temperature agarose gel and excised from the gel.
The gel slice is melted at 70?C., and the DNA is extracted twice
with an equal volume of Tris-buffered phenol, precipitated with
EtOH, and resuspended in 10?1 H.sub.2O.
[0134] The zkun10 cDNA is cloned into the EcoRV-AscI sites of a
modified pAdTrack-CMV (He, T-C. et al., Proc. Natl. Acad. Sci. USA
95:2509-2514, 1998). This construct contains the green fluorescent
protein (GFP) marker gene. The CMV promoter driving GFP expression
is replaced with the SV40 promoter, and the SV40 polyadenylation
signal is replaced with the human growth hormone polyadenylation
signal. In addition, the native polylinker is replaced with FseI,
EcoRV, and AscI sites. This modified form of pAdTrack-CMV is named
pZyTrack. Ligation is performed using a commercially available DNA
ligation and screening kit (Fast-Link.RTM. kit; Epicentre
Technologies, Madison, Wis.). Clones containing zalpha51 are
identified by digestion of mini prep DNA with FseI and AscI. In
order to linearize the plasmid, approximately 5 .mu.g of the
resulting pZyTrack zkun10 plasmid is digested with PmeI.
Approximately 1 .mu.g of the linearized plasmid is cotransformed
with 200 ng of supercoiled pAdEasy (He et al., ibid.) into E. coli
BJ5183 cells (He et al., ibid.). The co-transformation is done
using a Bio-Rad Gene Pulser at 2.5 kV, 200 ohms and 25 .mu.Fa. The
entire co-transformation mixture is plated on 4 LB plates
containing 25 .mu.g/ml kanamycin. The smallest colonies are picked
and expanded in LB/kanamycin, and recombinant adenovirus DNA is
identified by standard DNA miniprep procedures. The recombinant
adenovirus miniprep DNA is transformed into E. coli DH10B.TM. T
competent cells, and DNA is prepared using a Maxi Kit (Qiagen,
Inc.) according to kit instructions.
[0135] Approximately 5 .mu.g of recombinant adenoviral DNA is
digested with PacI enzyme (New England Biolabs) for 3 hours at
37.degree. C. in a reaction volume of 100 .mu.l containing 20-30U
of Pacd. The digested DNA is extracted twice with an equal volume
of phenol/chloroform and precipitated with ethanol. The DNA pellet
is resuspended in 10 .mu.l distilled water. A T25 flask of QBI-293A
cells (Quantum Biotechnologies, Inc. Montreal, Qc. Canada),
inoculated the day before and grown to 60-70% confluence, is
transfected with the Pacd digested DNA. The PacI-digested DNA is
diluted up to a total volume of 50 .mu.l with sterile HBS (150mM
NaCl, 2OmM HEPES). In a separate tube, 20 .mu.l of 1 mg/ml
N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium salts
(DOTAP) (Boehringer Mannheim, Indianapolis, Ind.) is diluted to a
total volume of 100 .mu.l with HBS. The DNA is added to the DOTAP,
mixed gently by pipeting up and down, and left at room temperature
for 15 minutes. The media is removed from the 293A cells and washed
with 5 ml serum-free minimum essential medium (MEM) alpha
containing 1 mM sodium pyruvate, 0.1 mM MEM non-essential amino
acids, and 25mM HEPES buffer (reagents obtained from Life
Technologies, Gaithersburg, Md.). 5 ml of serum-free MEM is added
to the 293A cells and held at 37.degree. C. The DNA/lipid mixture
is added drop-wise to the T25 flask of 293A cells, mixed gently,
and incubated at 37.degree. C. for 4 hours. After 4 hours the media
containing the DNA/lipid mixture is aspirated off and replaced with
5 ml complete MEM containing 5% fetal bovine serum. The transfected
cells are monitored for GFP expression and formation of foci (viral
plaques).
[0136] Seven days after transfection of 293A cells with the
recombinant adenoviral DNA, the cells express the GFP protein and
start to form foci (viral "plaques"). The crude viral lysate is
collected using a cell scraper to collect all of the 293A cells.
The lysate is transferred to a 50-ml conical tube. To release most
of the virus particles from the cells, three freeze/thaw cycles are
done in a dry ice/ethanol bath and a 37.degree. C. waterbath.
[0137] The crude lysate is amplified (Primary (1.degree.)
amplification) to obtain a working "stock" of zkun10 rAdV lysate.
Ten 10cm plates of nearly confluent (80-90%) 293A cells are set up
20 hours previously, 200 ml of crude rAdV lysate is added to each
10-cm plate, and the cells are monitored for 48 to 72 hours for CPE
(cytopathic effect) under the white light microscope and expression
of GFP under the fluorescent microscope. When all of the 293A cells
show CPE, this stock lysate is collected and freeze/thaw cycles
performed as described above.
[0138] A secondary (2.degree.) amplification of zkun10 rAdV is then
performed. Twenty 15-cm tissue culture dishes of 293A cells are
prepared so that the cells are 80-90% confluent. All but 20 ml of
5% MEM media is removed, and each dish is inoculated with 300-500
ml of the 1.degree. amplified rAdv lysate. After 48 hours the 293A
cells are lysed from virus production, the lysate is collected into
250-ml polypropylene centrifuge bottles, and the rAdV is
purified.
[0139] NP-40 detergent is added to a final concentration of 0.5% to
the bottles of crude lysate in order to lyse all cells. Bottles are
placed on a rotating platform for 10 minutes agitating as fast as
possible without the bottles falling over. The debris is pelleted
by centrifugation at 20,000.times.G for 15 minutes. The supernatant
is transferred to 250-ml polycarbonate centrifuge bottles, and 0.5
volume of 20% PEG8000/2.5 M NaCl solution is added. The bottles are
shaken overnight on ice. The bottles are centrifuged at
20,000.times.G for 15 minutes, and the supernatant is discarded
into a bleach solution. Using a sterile cell scraper, the white,
virus/PEG precipitate from 2 bottles is resuspended in 2.5 ml PBS.
The resulting virus solution is placed in 2-ml microcentrifuge
tubes and centrifuged at 14,000.times.G in the microcentrifuge for
10 minutes to remove any additional cell debris. The supernatant
from the 2-ml microcentrifuge tubes is transferred into a 15-ml
polypropylene snapcap tube and adjusted to a density of 1.34 glml
with CsCl. The solution is transferred to 3.2-ml, polycarbonate,
thick-walled centrifuge tubes and spun at 348,000.times.G for 3-4
hours at 25?C. The virus forms a white band. Using wide-bore
pipette tips, the virus band is collected.
[0140] A commercially available ion-exchange columns (e.g., PD-10
columns prepacked with Sephadex.RTM. G-25M; Pharmacia Biotech,
Piscataway, N.J.) is used to desalt the virus preparation. The
column is equilibrated with 20 ml of PBS. The virus is loaded and
allowed to run into the column. 5 mnl of PBS is added to the
column, and fractions of 8-10 drops are collected. The optical
densities of 1:50 dilutions of each fraction are determined at 260
nm on a spectrophotometer. Peak fractions are pooled, and the
optical density (OD) of a 1:25 dilution is determined. OD is
converted to virus concentration using the formula: (OD at
260nm)(25)(1.1.times.10.sup.12)=virions/ml.
[0141] To store the virus, glycerol is added to the purified virus
to a final concentration of 15%, mixed gently but effectively, and
stored in aliquots at -80?C.
[0142] A protocol developed by Quantum Biotechnologies, Inc.
(Montreal, Canada) is followed to measure recombinant virus
infectivity. Briefly, two 96-well tissue culture plates are seeded
with 1.times.10.sup.4293A cells per well in MEM containing 2% fetal
bovine serum for each recombinant virus to be assayed. After 24
hours 10-fold dilutions of each virus from 1.times.10.sup.-2 to
1.times.10 hu -14 are made in MEM containing 2% fetal bovine serum.
100 .mu.l of each dilution is placed in each of 20 wells. After 5
days at 37.degree. C., wells are read either positive or negative
for CPE, and a value for "Plaque Forming Units/ml" (PFU) is
calculated.
Example 5
Activity Assays
[0143] A. Trypsin Inhibitory Activity Assay on Mammalian Cell
Culture Supernatants
[0144] Conditioned media from cells expressing Kunitz-type
inhibitors is assayed for trypsin inhibitor activity. For each
clone, 20-100 .mu.l of conditioned medium is added to a solution
containing 2.4 .mu.g/ml trypsin (Worthington Biochemical, Freehold,
N.J.) in 100 mM NaCl, 50 mM Tris (pH 7.4) to give a final volume of
300 .mu.l. The reactions are incubated at 23.degree. C. for 30
minutes after which 20 .mu.l of 10 mM chromogenic substrate S-2251
(D-Val-Leu-Lys-Nan; Chromogenix, AB, Molndal, Sweden) is added to a
final concentration of 0.6 mM. The residual trypsin activity is
measured by absorbance at 405 nm.
[0145] B. Activity Assay on Yeast Culture Supernatants
[0146] Trypsin inhibitory activity is measured on the spent media
from cultures of yeast transformants described in Example 3 by
diluting 3.2 .mu.l of each spent medium sample with 80 .mu.l of
assay buffer (50 mM Tris HCl, pH 7.4, 100 mM NaCl, 2 mM CaCl.sub.2,
0.1% w/v PEG 20,000). The diluted supernatant is added to 80 ml of
133 nM bovine trypsin (Novo Nordisk A/S, Copenhagen, DK) diluted in
assay buffer, and the mixture is incubated for 10 minutes at room
temperature. After incubation, 100 ml of 1.8 mM peptidyl
nitroanilide substrate S2251 (D-Val-Leu-Lys-Nan; Kabi) diluted in
assay buffer is added to each sample, and the samples are incubated
with the substrate for 30 minutes. Trypsin inhibitory activity is
indicated by a colorless solution. A control reaction, which
results in a yellow solution, is produced by a supernatant from a
yeast strain not expressing any Kunitz-type inhibitor.
Example 6
Purification of Kunitz-Type Inhibitors
[0147] A. Purification of Kunitz-Type Inhibitors from Transfected
Mammalian Cell Culture Supernatants
[0148] zkun10 is purified from conditioned medium by sequential
application of heparin agarose, MONO Q, MONO S and SUPEROSE 12
chromatography as described in more detail below. Conditioned
serum-free media is adjusted to pH 7.5 with 1 N NaOH and filtered
through a 0.22-.mu.m filter. A 2.6.times.35 cm heparin sepharose
column (Pharmacia Biotech Inc., Piscataway, N.J.) is equilibrated
at 4.degree. C. with Buffer A (50 mM Tris-HCl (pH 7.5), 10%
glycerol). The filtered media is applied to the equilibrated column
at a flow rate of 3 ml/min. Following sample application, the
column is washed with Buffer A containing 0.2 M NaCl. zkun10
activity, as judged by its ability to inhibit trypsin is eluted
from the column with Buffer A containing 1 M NaCl. The eluent from
the heparin sepharose column is dialyzed at 4.degree. C. against 25
mM Tris-HCl (pH 7.5), 10% glycerol. The retentate is subjected to
FPLC (Pharmacia Biotech Inc.) on a 5.times.50 mm column containing
an anion exchanger with quaternary amine groups crosslinked to a
beaded hydrophylic resin such as a MONO Q (MONO Q HR 5/5; Pharmacia
Biotech Inc., Piscataway, N.J.) or the like that has been
equilibrated with 25 mM Tris-HCl (pH 7.5), 10% glycerol at room
temperature. zkun10 is eluted from the column in a linear NaCl
gradient (from 0-0.5 M NaCl) at a flow rate of 1 ml/min. The zkun10
fractions are pooled and dialyzed against 25 mM sodium citrate (pH
5.0), 10% glycerol. The retentate is then subjected to FPLC at room
temperature on a 5.times.50 mm column containing a cation exchanger
with charged sulfonic groups coupled to a beaded hydrophylic resin
such as MONO S (MONO S HR 5/5, Pharmacia Biotech Inc.) or the like
at a flow rate of 0.5 ml/min. zkun10 activity is eluted from the
MONO S column with a gradient elution from 25 mM sodium citrate (pH
5.0), 10% glycerol to 25 mM Tris-HCI (pH 7.5), 10% glycerol, 1 M
NaCl. Fractions containing zkun10 activity are pooled and
concentrated to approximately 1 ml by ultrafiltration. The
concentrated samples are subjected to FPLC across a cross-linked
agarose gel filtration matrix having a porosity suitable for the
separation of proteins from 1.times.10.sup.3 to 3.times.10.sup.5 MW
such as SUPEROSE 12 (Pharmacia Biotech Inc., Piscataway, N.J.) or
the like at room temperature in 50 mM Tris-HCl (pH 7.5), 100 mM
NaCl. Fractions eluted from the FPLC with zkun10 activity are
subjected to SDS-PAGE, and pure fractions are pooled and stored at
-80.degree. C.
[0149] B. Purification of Kunitz-Type Inhibitors from Yeast Culture
Supernatants
[0150] Kunitz-type inhibitors are purified from yeast culture
supernatants essentially as described by Norris et al. (ibid.;
which is incorporated herein by reference). Selected transformants
are grown in 10 liters of YEPD for approximately 40 hours at
30.degree. C. until an OD.sub.600 of approximately 25 has been
reached. The culture is centrifuged, and the supernatant is
decanted.
[0151] For purification, a 300 ml-1000 ml aliquot of supernatant is
adjusted to pH 2.3 and applied to a column holding 8 ml of
S-Sepharaose (Pharmacia-LKB Biotechnology AS, Alleroed, Denmark)
that has been previously equilibrated with 20 mM Bicine, pH 8.7
(Sigma Chemical Co., St. Louis, Mo.). After the column has been
extensively washed with 20 mM Bicine, pH 8.7, the Kunitz-type
inhibitor is eluted with 30 ml of 20 mM Bicine, pH 8.7 containing 1
M NaCl. The eluted material is desalted by application to a
Sephadex G-25 column (Pharmacia-LKB Biotechnology AS, Alleroed,
Denmark; 2.5.times.30 cm) that has been equilibrated with 20 mM
NH.sub.4HCO.sub.3, pH 7.8. The Kunitz-type inhibitor is eluted with
20 mM NH.sub.4HCO.sub.3, pH 7.8.
[0152] The Kunitz-type inhibitor is further purified and
concentrated by chromatography on a Mono S column (Pharmacia-LKB
Biotechnology AS, Alleroed, Denmark; 0.5.times.5 cm) equilibrated
with 20 mM Bicine, pH 8.7. After washing with the equilibration
buffer at 2 ml/min for 10 minutes, gradient elution of the
Kunitz-type inhibitor is carried out over twelve minutes at 1
ml/min from 0-0.6 M NaCl in the equilibration buffer. Peak samples
are pooled, and the Kunitz-type inhibitor is purified using reverse
phase HPLC on a Vydac 214TP510 column (Mikro-lab, Aarhus, Denmark;
1.0.times.25 cm) with a gradient elution at 4 ml/min from 5% A
(0.1% trifluoroacetic acid (TFA) in water) to 45% B (0.7% TFA in
acetonitrile) in 20 minutes. The purified product in lyophilized in
water, and inhibitor activity is measured.
[0153] Kunitz inhibitor activity is measured using the method
essentially described by Norris et al. (ibid.). Briefly, various
fixed concentrations of the Kunitz-type inhibitor are incubated in
the presence of 0.24 .mu.g/ml of porcine trypsin (Novo Nordisk A/S,
Bagsvaerd, Denmark), 12.8 CU/I human plasmin (Kabi, Stockholm,
Sweden) or 0.16 nkat/ml human plasma kallikrein (Kabi) in 100 mM
NaCl, 50 mM Tris HCI, pH 7.4. After a 30 minute incubation the
residual enzymatic activity is measured by the cleavage of a
substrate solution containing 0.6 mM of either of the chromogenic
peptidyl nitroanilide trypsin/plasmin substrates S2251
(D-Val-Leu-Lys-Nan; Kabi) or S2302 (D-Pro-Phe-Arg-Nan; Kabi) in
assay buffer. The samples are incubated for 30 minutes after which
the absorbance of each sample is measured at 405 nm. Plasmin or
trypsin activity is measured as a decrease in absorbance at 405 nm.
From the results, the apparent inhibition constant Ki is
calculated.
Example 7
Effect of Recombinant zkun10 on the Amydolytic Activities of Human
Thrombin, and Human Factor XA
[0154] A. Thrombin Amidolytic Activity Assay
[0155] The ability of recombinant zkun10 to inhibit the amidolytic
activity of human thrombin is determined by a colometric assay
using human thrombin (prepared as described by Pedersen, et al., J.
Biol. Chem. 265: 16786-16793, 1990; which is incorporated by
reference herein in its entirety) and various concentrations of
recombinant zkun10. The assay is set up in a microtiter plate
format. Reactions of 200 .mu.l are prepared in the wells of the
microtiter plate. The reaction mixtures contain various
concentrations of recombinant zkun10 and 20 nM human thrombin in 50
mM Tris-HCl (pH 7.5), 0.1% BSA, 5 mM CaCI.sub.2. The reactions are
incubated at 37.degree. C. for 15 minutes. Following incubation, 50
.mu.l of 10 mM the chromogenic substrate S-2238
(H-D-Phe-Pip-Arg-p-nitroanilide- , Chromogenix, AB, Molndal,
Sweden) is added to each well. The absorbance at 405 nm is
determined in a kinetic microplate reader (Model UVMAX, Molecular
Devices).
[0156] B. Human Factor Xa Amidolytic Assay
[0157] The ability of zkun10 to inhibit the amidolytic activity of
factor Xa is determined by a colorimetric assay as described above
using 20 nM human factor Xa (prepared as described by Kondo, and
Kisiel, Blood 70, 1947-1954, 1987; which is incorporated by
reference herein in its entirety) in place of the 20 nM human
thrombin described above. The reactions are set up and incubated as
described above replacing the human thrombin with human factor Xa.
Following incubation, 50 ml of 10 mM of the chromogenic substrate
S-2222 (Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide, Chromogenix, AB,
Molndal, Sweden) is added to each well. The absorbance at 405 nm is
determined in a kinetic microplate reader (Model UVMAX, Molecular
Devices).
[0158] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
6 1 836 DNA Homo sapiens CDS (1)...(333) 1 att ttt cta gaa gag aag
aga aaa gac atc aca aca tct ata act cag 48 Ile Phe Leu Glu Glu Lys
Arg Lys Asp Ile Thr Thr Ser Ile Thr Gln 1 5 10 15 caa gaa gca ctt
gaa aat tat gaa aat aac aaa tat gac att gaa gaa 96 Gln Glu Ala Leu
Glu Asn Tyr Glu Asn Asn Lys Tyr Asp Ile Glu Glu 20 25 30 aat gaa
caa gaa aca cca gca aaa caa aaa gaa act aga aaa gaa ata 144 Asn Glu
Gln Glu Thr Pro Ala Lys Gln Lys Glu Thr Arg Lys Glu Ile 35 40 45
aat gca gac act acc tat ggt cct tgt tcc atg gat cca atg gaa ggc 192
Asn Ala Asp Thr Thr Tyr Gly Pro Cys Ser Met Asp Pro Met Glu Gly 50
55 60 gag tgt cag gat cac acc ctg aag tgg cat tac aac aag gag gaa
cgg 240 Glu Cys Gln Asp His Thr Leu Lys Trp His Tyr Asn Lys Glu Glu
Arg 65 70 75 80 gtt tgc cag cag ttc tgg tgt ggc agc tgt ggc ggc aat
gcc aac cgg 288 Val Cys Gln Gln Phe Trp Cys Gly Ser Cys Gly Gly Asn
Ala Asn Arg 85 90 95 ttt gaa acc aag gaa gaa tgt gag gct tgg tgt
gtc cca ata cag 333 Phe Glu Thr Lys Glu Glu Cys Glu Ala Trp Cys Val
Pro Ile Gln 100 105 110 taacagtaca agcagagccc tgttactgtt aaaggcagag
cttttaatgc tgatgaaatg 393 gagattacca gggctgaggc aggacctcac
agctcagaag tgacagccca ttccaacacc 453 ttggacatca gattcctaaa
cgtctgaatg ttttcacgcc aacaaggact tgggccagat 513 gatttgtgac
ttgaggactg aattctaata gttaaaaaag taactgaaag atatttaaat 573
gaattagaac ggaatgaaaa ataaacttga acttataata ttattttaaa atttgggggt
633 gctatgtagc aaaataaaaa tcagtgtaag cagtgagaaa aacctaattc
agaaatgaat 693 cgaaacttgg tttgtttttt tcaccaccag agaataggga
aatattagtc aaagagaggg 753 catggaagaa gggacatcta atgtgaacga
acttcatact tactacttaa tgtagataaa 813 taaaggcatt ctttattaaa tca 836
2 111 PRT Homo sapiens 2 Ile Phe Leu Glu Glu Lys Arg Lys Asp Ile
Thr Thr Ser Ile Thr Gln 1 5 10 15 Gln Glu Ala Leu Glu Asn Tyr Glu
Asn Asn Lys Tyr Asp Ile Glu Glu 20 25 30 Asn Glu Gln Glu Thr Pro
Ala Lys Gln Lys Glu Thr Arg Lys Glu Ile 35 40 45 Asn Ala Asp Thr
Thr Tyr Gly Pro Cys Ser Met Asp Pro Met Glu Gly 50 55 60 Glu Cys
Gln Asp His Thr Leu Lys Trp His Tyr Asn Lys Glu Glu Arg 65 70 75 80
Val Cys Gln Gln Phe Trp Cys Gly Ser Cys Gly Gly Asn Ala Asn Arg 85
90 95 Phe Glu Thr Lys Glu Glu Cys Glu Ala Trp Cys Val Pro Ile Gln
100 105 110 3 333 DNA Artificial Sequence degenerate sequence 3
athttyytng argaraarmg naargayath acnacnwsna thacncarca rgargcnytn
60 garaaytayg araayaayaa rtaygayath gargaraayg arcargarac
nccngcnaar 120 caraargara cnmgnaarga rathaaygcn gayacnacnt
ayggnccntg ywsnatggay 180 ccnatggarg gngartgyca rgaycayacn
ytnaartggc aytayaayaa rgargarmgn 240 gtntgycarc arttytggtg
yggnwsntgy ggnggnaayg cnaaymgntt ygaracnaar 300 gargartgyg
argcntggtg ygtnccnath car 333 4 51 PRT Artificial Sequence kunitz
motif 4 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Xaa 20 25 30 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa 35 40 45 Xaa Xaa Cys 50 5 1747 DNA Homo sapiens
CDS (1)...(1248) 5 atg gat gga acc aac aga ttt tac ttg tac gtc tgg
gag aca gag cgc 48 Met Asp Gly Thr Asn Arg Phe Tyr Leu Tyr Val Trp
Glu Thr Glu Arg 1 5 10 15 cag cag gat gtg gag cac gtg gcc cgc tgt
att ctc tgc tat gac aaa 96 Gln Gln Asp Val Glu His Val Ala Arg Cys
Ile Leu Cys Tyr Asp Lys 20 25 30 tgc aga cca gac cca gaa tgc ccg
gct ggc acg ccg ggg ccc cag gag 144 Cys Arg Pro Asp Pro Glu Cys Pro
Ala Gly Thr Pro Gly Pro Gln Glu 35 40 45 gtg gac gtg gac ttg gta
ttt gtg gtg gac agc tcc tat gga gtg gat 192 Val Asp Val Asp Leu Val
Phe Val Val Asp Ser Ser Tyr Gly Val Asp 50 55 60 gcc gac gtg tac
cgc ggg tct ttg agt cta gcg gac gcc gcg cta gaa 240 Ala Asp Val Tyr
Arg Gly Ser Leu Ser Leu Ala Asp Ala Ala Leu Glu 65 70 75 80 gac ctg
gag gtg gct gag cag ccg ggc gcg tcc cac cgt ggg gcg cgt 288 Asp Leu
Glu Val Ala Glu Gln Pro Gly Ala Ser His Arg Gly Ala Arg 85 90 95
gtg gcc ctg gtg acg cac acg aca ccc aac ttc tgg ccg ggg ctt cca 336
Val Ala Leu Val Thr His Thr Thr Pro Asn Phe Trp Pro Gly Leu Pro 100
105 110 ctt gac cac cta tgg caa ccg gaa gca gat gca gag aca tgt gcg
cga 384 Leu Asp His Leu Trp Gln Pro Glu Ala Asp Ala Glu Thr Cys Ala
Arg 115 120 125 ggc ttc agc cgc ccc tta cag gga acc gcc ccc cct ggc
cac gcc ctg 432 Gly Phe Ser Arg Pro Leu Gln Gly Thr Ala Pro Pro Gly
His Ala Leu 130 135 140 gag tgg acg ctg gag aat gtg ctc ctg gca gcc
cct cgg ccg cgg aag 480 Glu Trp Thr Leu Glu Asn Val Leu Leu Ala Ala
Pro Arg Pro Arg Lys 145 150 155 160 gca caa gtc ctc ttc gcc atc gtg
gcc agc gag aca agt agc tgg gac 528 Ala Gln Val Leu Phe Ala Ile Val
Ala Ser Glu Thr Ser Ser Trp Asp 165 170 175 agg gag aag cta tgg act
ctg tcc ctg gag gcc aaa tgc aag ggc att 576 Arg Glu Lys Leu Trp Thr
Leu Ser Leu Glu Ala Lys Cys Lys Gly Ile 180 185 190 acc ctc ttt gtg
ctg gcc ttg ggt ccg ggt gtg ggg acc cat gag cta 624 Thr Leu Phe Val
Leu Ala Leu Gly Pro Gly Val Gly Thr His Glu Leu 195 200 205 gcc gag
cta gcc gag ctg gtc agt gct ccc tct gag cag cat cta ctg 672 Ala Glu
Leu Ala Glu Leu Val Ser Ala Pro Ser Glu Gln His Leu Leu 210 215 220
cgc cta caa ggg gtc tca gag cca gag gtt aac tac gct cag gga ttc 720
Arg Leu Gln Gly Val Ser Glu Pro Glu Val Asn Tyr Ala Gln Gly Phe 225
230 235 240 act cgg gcc ttc ctg aac ctc cta aaa agt ggg aca aac cag
tac cca 768 Thr Arg Ala Phe Leu Asn Leu Leu Lys Ser Gly Thr Asn Gln
Tyr Pro 245 250 255 ccc cca gag ctc act gaa gaa tgt ggg ggc cta cac
cgt ggg gac act 816 Pro Pro Glu Leu Thr Glu Glu Cys Gly Gly Leu His
Arg Gly Asp Thr 260 265 270 gtg ctg caa tta gtc aca cct gtc aac agg
ttg ccc agg cac cag ttt 864 Val Leu Gln Leu Val Thr Pro Val Asn Arg
Leu Pro Arg His Gln Phe 275 280 285 ggt atg tct ggc ttg gct gat gat
ttg gaa gca ctt gaa gca aca ggc 912 Gly Met Ser Gly Leu Ala Asp Asp
Leu Glu Ala Leu Glu Ala Thr Gly 290 295 300 att ttt cta gaa gag aag
aga aaa gac atc aca aca tct ata act cag 960 Ile Phe Leu Glu Glu Lys
Arg Lys Asp Ile Thr Thr Ser Ile Thr Gln 305 310 315 320 caa gaa gca
ctt gaa aat tat gaa aat aac aaa tat gac att gaa gaa 1008 Gln Glu
Ala Leu Glu Asn Tyr Glu Asn Asn Lys Tyr Asp Ile Glu Glu 325 330 335
aat gaa caa gaa aca cca gcc aaa caa aca gca act aga aaa gaa ata
1056 Asn Glu Gln Glu Thr Pro Ala Lys Gln Thr Ala Thr Arg Lys Glu
Ile 340 345 350 aat gca gac act acc tat ggt cct tgt tcc atg gat cca
atg gaa ggc 1104 Asn Ala Asp Thr Thr Tyr Gly Pro Cys Ser Met Asp
Pro Met Glu Gly 355 360 365 gag tgt cag gat cac acc ctg aag tgg cat
tac aac aag gag gaa cgg 1152 Glu Cys Gln Asp His Thr Leu Lys Trp
His Tyr Asn Lys Glu Glu Arg 370 375 380 gtt tgc cag cag ttc tgg tgt
ggc agc tgt ggc ggc aat gcc aac cgg 1200 Val Cys Gln Gln Phe Trp
Cys Gly Ser Cys Gly Gly Asn Ala Asn Arg 385 390 395 400 ttt gaa acc
aag gaa gaa tgt gag gct tgg tgt gtc cca ata cag taa 1248 Phe Glu
Thr Lys Glu Glu Cys Glu Ala Trp Cys Val Pro Ile Gln * 405 410 415
cagtacaagc agagccctgt tactgttaaa ggcagagctt ttaatgctga tgaaatggag
1308 attaccaggg ctgaggcagg acctcacagc tcagaagtga cagcccattc
caacaccttg 1368 gacatcagat tcctaaacgt ctgaatgttt tcacgccaac
aaggacttgg gccagatgat 1428 ttgtgacttg aggactgaat tctaatagtt
aaaaaagtaa ctgaaagata tttaaatgaa 1488 ttagaacgga atgaaaaata
aacttgaact tataatatta ttttaaaatt tgggggtgct 1548 atgtagcaaa
ataaaaatca gtgtaagcag tgagaaaaac ctaattcaga aatgaatcga 1608
aacttggttt gtttttttca ccaccagaga atagggaaat attagtcaaa gagagggcat
1668 ggaagaaggg acatctaatg tgaacgaact tcatacttac tacttaatgt
agataaataa 1728 aggcattctt tattaaatc 1747 6 415 PRT Homo sapiens 6
Met Asp Gly Thr Asn Arg Phe Tyr Leu Tyr Val Trp Glu Thr Glu Arg 1 5
10 15 Gln Gln Asp Val Glu His Val Ala Arg Cys Ile Leu Cys Tyr Asp
Lys 20 25 30 Cys Arg Pro Asp Pro Glu Cys Pro Ala Gly Thr Pro Gly
Pro Gln Glu 35 40 45 Val Asp Val Asp Leu Val Phe Val Val Asp Ser
Ser Tyr Gly Val Asp 50 55 60 Ala Asp Val Tyr Arg Gly Ser Leu Ser
Leu Ala Asp Ala Ala Leu Glu 65 70 75 80 Asp Leu Glu Val Ala Glu Gln
Pro Gly Ala Ser His Arg Gly Ala Arg 85 90 95 Val Ala Leu Val Thr
His Thr Thr Pro Asn Phe Trp Pro Gly Leu Pro 100 105 110 Leu Asp His
Leu Trp Gln Pro Glu Ala Asp Ala Glu Thr Cys Ala Arg 115 120 125 Gly
Phe Ser Arg Pro Leu Gln Gly Thr Ala Pro Pro Gly His Ala Leu 130 135
140 Glu Trp Thr Leu Glu Asn Val Leu Leu Ala Ala Pro Arg Pro Arg Lys
145 150 155 160 Ala Gln Val Leu Phe Ala Ile Val Ala Ser Glu Thr Ser
Ser Trp Asp 165 170 175 Arg Glu Lys Leu Trp Thr Leu Ser Leu Glu Ala
Lys Cys Lys Gly Ile 180 185 190 Thr Leu Phe Val Leu Ala Leu Gly Pro
Gly Val Gly Thr His Glu Leu 195 200 205 Ala Glu Leu Ala Glu Leu Val
Ser Ala Pro Ser Glu Gln His Leu Leu 210 215 220 Arg Leu Gln Gly Val
Ser Glu Pro Glu Val Asn Tyr Ala Gln Gly Phe 225 230 235 240 Thr Arg
Ala Phe Leu Asn Leu Leu Lys Ser Gly Thr Asn Gln Tyr Pro 245 250 255
Pro Pro Glu Leu Thr Glu Glu Cys Gly Gly Leu His Arg Gly Asp Thr 260
265 270 Val Leu Gln Leu Val Thr Pro Val Asn Arg Leu Pro Arg His Gln
Phe 275 280 285 Gly Met Ser Gly Leu Ala Asp Asp Leu Glu Ala Leu Glu
Ala Thr Gly 290 295 300 Ile Phe Leu Glu Glu Lys Arg Lys Asp Ile Thr
Thr Ser Ile Thr Gln 305 310 315 320 Gln Glu Ala Leu Glu Asn Tyr Glu
Asn Asn Lys Tyr Asp Ile Glu Glu 325 330 335 Asn Glu Gln Glu Thr Pro
Ala Lys Gln Thr Ala Thr Arg Lys Glu Ile 340 345 350 Asn Ala Asp Thr
Thr Tyr Gly Pro Cys Ser Met Asp Pro Met Glu Gly 355 360 365 Glu Cys
Gln Asp His Thr Leu Lys Trp His Tyr Asn Lys Glu Glu Arg 370 375 380
Val Cys Gln Gln Phe Trp Cys Gly Ser Cys Gly Gly Asn Ala Asn Arg 385
390 395 400 Phe Glu Thr Lys Glu Glu Cys Glu Ala Trp Cys Val Pro Ile
Gln 405 410 415
* * * * *