Note: Descriptions are shown in the official language in which they were submitted.
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Description
KUNITZ DOMAIN POLYPEPTIDE ZKUN6
BACKGROUND OF THE INVENTION
In animals, proteinases are important in wound healing,
extracellular matrix destruction, tissue reorganization, and in cascades
leading
1o 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.
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, kalfikrein, plasmin, coagulation factors Xla and IXa,
and cathepsin G. These inhibitors thus regulate a variety of physiological
2 0 processes, including blood coagulation, fibrinolysis, and inflammation.
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
3 0 a modified inhibitor that is cleaved at the scissile bond of the binding
loop.
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
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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., Peterson 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).
Proteinase inhibitors comprising one or more Kunitz domains
include tissue factor pathway inhibitor (TFPI), tissue factor pathway
inhibitor 2
(TFPI-2), amyloid ~-protein precursor (A~iPP), 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 Vlla, plasmin, and cathepsin G; the second domain inhibits
factor Xa, trypsin, and chymotrypsin; and the third domain has no known
activity (Peterson et al., ibid.). TFPI-2 has been shown to be an inhibitor of
the
amidolytic and proteolytic activities of human factor Vlla-tissue factor
complex,
factor Xla, plasma kallikrein, and plasmin (Sprecher et al., Proc. Natl. Acad.
Sci. USA 91:3353-3357, 1994; Peterson et al., Biochem. 35:266-272, 1996).
The ability of TFPI-2 to inhibit the factor Vlla-tissue factor complex and its
relatively high levels of transcription in umbilical vein endothelial cells,
2 o 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 plamin, 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.
3 o Surg. 55:1033-1041, 1993; W achtfogel 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
3 5 Kunitz domains of bikunin have been expressed and shown to be potent
inhibitors of trypsin, chymotrypsin, plasmin, factor Xla, and tissue and
plasma
kallikrein (Detaria et al., ibid.).
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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.
SUMMARY OF THE INVENTION
It is an aspect of the present invention to provide novel Kunitz
inhibitor proteins and compositions comprising the proteins. It is another
aspect of the invention to provide materials and methods for making the Kunitz
inhibitor proteins. It is a further aspect of the invention to provide
antibodies
that specifically bind to the Kunitz inhibitor proteins.
Within one aspect, the invention provides an isolated protein
comprising a sequence of amino acid residues as shown in SEQ ID N0:3,
wherein the sequence is at least 80% identical to residues 6 through 56 of
SEQ ID N0:2. Within one embodiment, the protein is from 51 to 81 amino acid
residues in length. Within another embodiment, the sequence is at least 90%
identical to residues 6 through 56 of SEQ ID N0:2. Within another
embodiment, the sequence consists of residues 6 through 56 of SEQ ID N0:2.
Within other embodiments, the protein is from 51 to 67 residues in length,
preferably from 55 to 62 residues in length. Within an additional embodiment,
the protein further comprises an affinity tag. Suitable affinity. tags include
maltose binding protein, polyhistidine, and Glu-Tyr-Met-Pro-Met-Glu (SEQ ID
2 5 N0:6).
Within a second aspect, the invention provides an expression
vector comprising the following operably linked elements: (a) a transcription
promoter; (b) a DNA segment encoding a protein as disclosed above; and (c) a
transcription terminator. Within one embodiment, the expression vector further
3 0 comprises a secretory signal sequence operably linked to the DNA segment.
_ Within a third aspect, the invention provides a. cultured cell
containing an expression vector as disclosed above, wherein the cell
expresses the DNA segment. Within certain embodiments of the invention the
cell is a yeast cell or a mammalian cell.
35 Within a fourth aspect of the invention there is provided a method
of making a protein comprising culturing a cell as disclosed above under
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conditions whereby the DNA segment is expressed, and recovering the protein
encoded by the DNA segment.
Within a fifth aspect of the invention there is provided an
antibody that specifically binds to a protein of from 51 to 81 amino acid
residues comprising a sequence of amino acid residues as shown in SEQ ID '
N0:3, wherein the sequence is at least 80% identical to residues 6 through 56
'
of SEQ ID N0:2.
These and other aspects of the invention will become evident
upon reference to the following detailed description and the attached drawing.
BRIEF DESCRIPTION OF THE DRAWING
The attached drawing shows an amino acid sequence alignment
of a representative polypeptide of the present invention (SEQ ID N0:2),
designated "ZKUN6", with the sequence of the Kunitz domain of human alpha
3 type VI collagen (SEQ ID N0:5), designated "1 KNT'.
DETAILED DESCRIPTION OF THE INVENTION
Prior to setting forth the invention in detail, it may be helpful to
the understanding thereof to define the following terms:
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
(Niisson 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-
Gfu -'affinity tag (Glu-Tyr-Met-Pro-Met-Glu; SEQ ID N0:6) (Grussenmeyer et
al.,
Proa Natl. Acad Sci. USA 82:7952-4, 1985), substance P, FIagTM peptide
3 0 (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,
NJ).
3 5 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
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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.
5 The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides. Where the context allows,
these temps 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
1o poiypeptide is located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus of the complete
polypeptide.
A "complement" of a poiynucleotide 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' CCCGTGGAT 3'.
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
2 0 codons contain different triplets of nucleotides, but encode the same
amino
acid residue (i.e., GAU and GAC triplets each encode Asp).
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.
The term "expression vector° is used to denote a DNA molecule,
linear or circular, that comprises a segment encoding a pofypeptide of
interest
operably linked to additional segments that provide for its transcription.
Such
3 o 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. F~cpression vectors are
generally
derived from plasmid or viral DNA, or may contain elements of both.
The term "isolated", when applied to a polynucleotide, denotes
3 5 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.
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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).
An "isolated" polypeptide or protein is a polypeptide or protein
that is found in a condition other than its native environment, such as apart
1o from blood and animal tissue. In a preferred form, the isolated polypeptide
is
substantially free of other poiypeptides, 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.
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
2o proceeds through the coding segment to the terminator.
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.
A "polynucleotide" is a single-~ or double-stranded polymer of
deoxyribonucfeotide or ribonucieotide 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 ("ntn), 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
3 5 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
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polynucleotide molecule may not be paired. Such unpaired ends will in
general not exceed 20 nt in length.
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".
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
s0 sequences are commonly, but not always, found in the 5' non-coding regions
of genes.
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.
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 targer 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
2 5 pathway.
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
3 o 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.
Molecular weights and lengths of polymers determined by
35 imprecise analytical methods (e.g., gel electrophoresis) will be.
understood to
be approximate values. When such a value is expressed as "about" X or
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"approximately" X, the stated value of X will be understood to be accurate to
ti 0%.
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 "zkun6". The
zkun6
polypeptide sequence shown in SEQ ID N0:2 comprises this Kunitz domain,
which is bounded at the amino and carboxyl termini by cysteine residues at
positions 6 and 56, respectively.
Zkun6 has 45% residue identity with the 51-residue kunitz
1o domain in human alpha 3 type VI collagen (shown in SEQ ID N0:5). The
structure of the latter domain has been solved by X-ray crystallography and by
NMR (Amoux et al., J. Mol. Biol. 246:609-617, 1995; Sorensen et al.,
Biochemistry36:10439-10450, 1997). An alignment of zkun6 and the collagen
Kunitz domain (see the drawing) can be combined with a homology model of
zkun6 based on the X-ray structure to predict the function of certain residues
in
zkun6. Referring to SEQ ID N0:2, disulfide bonds are predicted to be formed
by paired cysteine residues Cys6 - Cys56; CyslS - Cys39; and Cys31 - Cys52.
The protease binding loop (P3-P4') is expected to comprise residues 14-20 of
SEQ fD N0:2 (Pro-Cys-Arg-Gly-Trp-Glu-Pro), with the P1 residue being Argl6,
and the P1' residue being GIyl7.
Functional derivatives of the zkun6 sequence, characterized by
one or more amino acid deletions, substitutions, insertions, or additions, are
also within the scope of the present invention. Such amino acid alterations
can be made within the zkun6 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 zkun6 Kunitz domain by reference to
the sequences of other Kunitz domains. SEQ ID N0:3 is a generalized Kunitz
domain sequence that shows allowable amino acid substitutions based on
such an alignment. The 51-residue sequence shown in SEQ ID N0:3
3 o conforms to the pattern:
C-X(8)-C-X(15)-C-X(7)-C-X(12)-C-X(3)-C -
wherein C denotes cysteine; X is any naturally occuring amino acid residue,
subject to the limitations set forth in the attached Sequence Listing for SEQ
ID
N0:3; and the numerals indicate the number of such variable residues. The
second cysteine residue is in the P2 position.
Within the present invention up to 20% of the amino acid
residues in the zkun6 Kunitz domain (residues 6 through 56 of SEQ ID N0:2)
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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 N0:3. The present invention thus provides a family of proteins comprising a
sequence of amino acid residues as shown in SEQ ID N0:3, wherein the
sequence is at least 80% identical to residues 6 through 56 of SEQ ID N0:2. It
is preferred that the proteins of the present invention comprise such a
sequence that is at least 85%, more preferably at least 90%, and most
preferably at least 95% identical to residues 6 through 56 of SEQ ID N0:2.
Percent sequence identity is determined by conventional
1o methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603-616,
1986,
and Henikoff and Henikoff, Proc. Nail. 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
"BLOSUM62u scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table
i5 1 (amino acids are indicated by the standard one-letter codes). The percent
identity is then calculated as:
Total number of identical matches
x 100
20 [length of the longer sequence plus the
number of gaps introduced into the longer
sequence in order to align the two sequences]
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). d'
r
1
'-N M
r 1
~ N N O
1 1
r' ~"~N N
a ' ' '
I~ r r ~tM N
1 1 1
(O'~ N N r ~ r
O N r r r T r
T Y r 1 1 1 1 1
m M r r C'~N N
O 1 1 , 1
I~ J ~ N N O ~ N '' N r- r
_ 1 ' 1 ' ' '
N M r O M N r M r M
1 , 1 1 1
O ~ M r N r'N ,_N N N CO
tCN ~fi~t N ~ ~ N p N N M M
1 1 , 1 1 1 1 1
tn N O N' M r O r C~N N
C'~t~ ,-, 1 , , , , 1
N N O 1 1 O
C'ON r O ~ T' r N r N
1 1 1 ~ 1 '
V O C'~d' ChM r r (~r N C'~r r N N r
t 1 1 1 1 . 1 ' ' 1 1 t 1 1
1
(O~ O N r r M ~' r C~ C'~r O r '~C'~ M
1 1 1 1 1 1 1 1 , 1
Z (G r C'~O O O r C'~C~ O N M N r O '~N M
1 1 1 1 , 1
~ O N ~ - O N O M N N T M N r r M N M
1 1 1
Q d' N N O r r O N r r r r N r r O C~CV O
r 1 1 1 1 ~ 1 1 1 ' ' 1 1 1
1
Q Z O U CWa C3Z - ~ Y ~ tia. cW- ~ >- >
OC
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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 8:2444, 1988) and by
Pearson (Meth. Enzymol. x:63, 1990). Briefly, FASTA first characterizes
sequence similarity by identifying regions shared by the query sequence
(e.g., SEQ ID N0:2) and a test sequence that have either the highest density
of identities (if the letup variable is 1 ) or pairs of identities (if
letup=2), without
considering conservative amino acid substitutions, insertions, or deletions.
The ten regions with the highest density of identities are then rescored by
1o 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 letup
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 acrd 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: letup=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
2 5 (ibid ).
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 letup value can range between one to six,
preferably from three to six, most preferably three, with other parameters set
3 o as default.
The proteins of the present invention can also comprise non-
naturally occurring amino acid residues. Non-naturally occurring amino acids
include, without limitation, traps-3-methylproline, 2,4-methanoproline, cis-4-
hydroxyproline, tran~4-hydroxyproline, N-methylglycine, allo-threonine,
3 5 methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,
nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid,
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dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tent 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, and
include cell-based, cell-free, and in vitro modification methods. See, for
example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al.,
Methods Fnzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993;
Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993; Turcatti et al., J.
Biol. Chem. 271:19991-8, 1996; Koide et al., Biochem. 33:7470-6, 1994; and
1o Wynn and Richards, Protein Sci. 2:395-403, 1993.
Additional polypeptides may be joined to the amino and/or
carboxyl termini of the zkun6 Kunitz domain (residues 6-56 of SEQ ID N0:2)
or a derivative of the zkun6 Kunitz domain as disclosed above. Particularly
preferred proteins in this regard include residues 1-59 of SEQ iD N0:2.
Amino and carboxyl extensions of the zkun6 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 zkun6 protein can
comprise residues 6-56 of SEQ ID N0:2 with amino- and carboxyl-terminal
dipeptides, wherein the individual amino acid residues of the dipeptides are
any amino acid residue except cysteine.
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 iinker 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 zkun6 portion and the affinity tag. Preferred
cieavage sites include thrombin cleavage sites and factor Xa cleavage sites.
For example, a zkun6 polypeptide of 59 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 N0:7)--zkun6,
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resulting in a polypeptide of approximately 439 residues. In a second
example, a zkun6 polypeptide of 81 residues can be fused to E. coli ~-
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,i 16 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 zkun6 proteins. In another example, a zkun6 Kunitz domain can
be expressed as a secreted protein comprising a carboxyl-terminal receptor
to 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-
tem~inal transmembrane domain--spacer polypeptide--Kunitz domain--amino-
terminal polypeptide. Many receptor . transmembrane domains and
polynucleotides encoding them are known in the art. The spacer potypeptide
will generally be at least about 50 amino acid residues in length, up to 200-
2 0 300 or more residues. The amino terminal polypeptide may be up to 300 or
more residues in length.
Also disclosed herein are polynucleotide molecules, including
DNA and RNA molecules, encoding zkun6 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 zkun6 protein is set forth in SEQ ID N0:1. DNA sequences encoding other
zkun6 proteins can be readily generated by those of ordinary skill in the art
based on the genetic code. Counterpart RNA sequences can be generated
3 o by substitution of U for T. Polynucleotides encoding zkun6 proteins and
complementary polynucleotides are useful in the production 'of zkun6 proteins
and for diagnostic and investigatory purposes.
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 N0:4 is a degenerate DNA
sequence that encompasses all DNAs that encode the zkun6 poiypeptide of
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SEQ ID N0:2. Those skilled in the art will recognize that the degenerate
sequence of SEQ ID N0:4 also provides all RNA sequences encoding SEQ _
ID N0:2 by substituting U for T. Thus, zkun6 polypeptide-encoding
polynucleotides comprising nucleotide 1 to nucleotide 177 of SEQ ID N0:4
and their respective RNA equivalents are contemplated by the present -- '
invention. Table 2 sets forth the one-letter codes used within SEQ ID N0:4 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,
1o and its complement R denotes A or G, A being complementary to T, and G
being complementary to C.
TABLE 2
Nucleotide Resolution Nucleotide Complement
A A T T
C C G G
G G C C
T T A A
R AIG Y CIT
Y CIT R AIG
M AIC K GIT
K GIT M ~AIC
S CIG S CIG
W AIT W AIT
H AICIT D AIGiT
B CIGIT V AICIG
V AICIG B CIGIT
D AIGIT H AICIT
N AICIGIT N AICIGIT
The degenerate codons used in SEQ ID N0:4, encompassing
all possible codons for a given amino acid, are set forth in Table 3.
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TABt~E 3
One
Amino Letter Codons Degenerate
Acid Code 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 pAY
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 . AqR
Met M ATG ATG
Ile I ATA ATC ATT ATH
Leu L CTA CTC CTG CTT TTA TTG YTN
Val V GTA GTC GTG GTT GTN
Phe F TTC TTT '~'~y
Tyr Y TAC TAT TAY
Trp W TGG . _ ~ TGG
Ter . TAA TAG TGA . TRR
AsnIAsp B . RAY
GIuIGIn Z SAR
Any X NNN
One of ordinary skill in the art will appreciate that some
ambiguity is introduced in determining a degenerate codon, representative of
5 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
phenylaianine and leucine. Thus, some polynucleotides encompassed by the
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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 !D N0:2. Variant
sequences can be readily tested for functionality as described herein.
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. 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. The degenerate codon sequence disclosed in SEQ ID N0:4 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
2 o disclosed herein.
It is preferred that zkun6 poiynucleotides hybridize to similar
sized regions of SEQ ID N0:1, or a sequence complementary thereto, under
stringent conditions. In general, stringent conditions are selected to be
about
5°C lower than the thermal melting point (Tm) for the specific sequence
at a
defined ionic strength and pH. The Tm 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°C.
3 o As previously noted, zkun6-encoding polynucleotides 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 zkun6 RNA. Such tissues and cells are identified by conventional
procedures, such as Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA
3 5 77:5201, 1980). Total RNA can be prepared using guanidine-HCI extraction
followed by isolation by centrifugation in a CsCI gradient (Chirgwin et al.,
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Biochemistry 1_x:52-94, 1979). Poly (A)+ RNA is prepared from total RNA
y using the method of Aviv and Leder (Prow Natl. Acad. Sci. USA ,9:1408-12,
1972}. Complementary DNA (cDNA) is prepared from poly(A)+ RNA using
known methods. In the alternative, genomic DNA can be isolated.
Polynucleotides encoding zkun6 polypeptides are then identified and isolated
by, for example, hybridization or PCR.
A full-length clone encoding zkun6 can be obtained by
conventional cloning procedures. Complementary DNA (cDNA) clones are
preferred, although for some applications (e.g., expression in transgenic
to 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 zkun6, receptor fragments, or other specific binding partners.
The polynucleotides of the present invention can also be
synthesized using automated equiprr~nt. The current method of choice is the
phosphoramidite method. If chemically synthesized double stranded DNA is
required for an application such as the synthesis of a gene or a gene
2 0 fragment, then each complementary strand is made separately. The
production of short genes (60 to 80 bp) is technically straightforward and can
be accomplished by synthesizing the complementary strands and then
annealing them. For the production of longer genes (>300 bp), however,
special strategies must be invoked, because the coupling efficiency of each
cycle during chemical DNA synthesis is seldom 100%. To overcome this
problem, synthetic genes (double-stranded} are assembled in modular form
from single-stranded fragments that are from 20 to 100 nucleotides in length.
Gene synthesis methods are well known in the art. See, for example, Glick
and Pastemak, Molecular Biotechnoloqy Principles & Applications of
3 o Recombinant DNA, ASM Press, Washington, D.C., 1994; Itakura et al., Annu.
Rev. Biochem. 53: 323-356, 1984; ~ and Climie et al., Proa Natl. Aced. Sci.
USA 87:633-637, 1990.
The zkun6 polynucleotide sequences disclosed herein can be
used to isolate counterpart poiynucleotides from other species (orthologs).
These orthologous polynucleotides can be used, inter alia, to prepare the
respective orthotogous proteins. These other species include, but are not
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limited to mammalian, avian, amphibian, reptile, fish, insect and other
vertebrate and invertebrate species. Of particular interest are zkun6
polynucleotides abd polypeptides from other mammalian species, including a-
murine, porcine, ovine, bovine, canine, feline, equine, and other primate
polypeptides. Orthologs of human zkun6 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 zkun6 as disclosed
herein. Suitable sources of mRNA can be identified by probing Northern blots
1o with probes designed from the sequences disclosed herein. A library is then
prepared from mRNA of a positive tissue or cell line. A zkun6-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 sequences. A cDNA can also be cloned using
the pofymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202),
using primers designed from the representative human zkun6 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 zkun6 polypeptide. Similar techniques
2o can also be applied to the isolation of genomic clones.
Those skilled in the art will recognize that the sequence
disclosed in SEQ ID N0:1 represents a single allele of human zkun6 and that
natural variation, including allelic variation and alternative splicing, is
expected to occur. Allelic variants of this sequence can be cloned by probing
cDNA or genomic libraries from different individuals according to standard
procedures. Allelic variants of the DNA sequence shown in SEQ ID NO:1,
including those containing silent mutations and those in which mutations
result in amino acid sequence changes, are within the scope of the present
invention, as are proteins which are allelic variants of SEQ ID N0:2. cDNAs
3 o generated from alternatively spliced mRNAs, which retain the proteinase
inhibiting activity of zkun6 are included within the scope of the present
invention, as are polypeptides encoded by such cDNAs and mRNAs. Allelic
variants and splice variants of these sequences can be cloned by probing
cDNA or genomic libraries from different individuals or tissues according to
3 5 standard procedures known in the art.
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Zkun6 proteins, including variants of wild-type zkun6, are tested
for activity in protease inhibition assays, a variety of which are known in
the
art. Preferred assays include those measuring inhib'ttion of trypsin,
chymotrypsin, plasmin, cathepsin G, and human leukocyte elastase. See, for
example, Peterson 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 corr~ound 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°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; is catculated. The inhibition of coagulation
factors (e.g., factor Vlla, factor Xa) can be . measured using chromogenic
substrates or in conventional coagulation assays (e.g., clotting time of
normal
2 o human plasma; Dennis et al., ibid.).
Zkun6 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
3 o 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. Zkunfi 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
3 5 transgenic expression.
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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 Backer et al.,
Meth.
5 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,
20 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 iigation or by homologous recombination with a co-transfected plasmid.
15 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
2o 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.
An aitemative 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
3o 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.
Transgenic mice, engineered to express a zkun6 gene, and
mice that exhibit a complete absence of zkun6 gene function, referred to as
"knockout mice" (Snouwaert et ai., Science 257:1083, 1992), can also be
generated (towel) et al., Nature 366:740-742, 1993). These mice are
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employed to study the zkun6 gene and the encoded protein in an in vivo
system. Transgenic mice are particularly useful for investigating the role of
zkun6 proteins in early development because they allow the identification of
developmental abnormalities or blocks resulting from the over- or
underexpression of a specific factor.
The zkun6 polypeptides of the present invention, including fuN-
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, NY, 1989, and Ausubel et al., eds.,
Current Protocols in Molecular Biology, John. Wiley and Sons, Inc., NY, 1987.
In general, a DNA sequence encoding a zkun6 polypeptide is
operably linked to other genetic elements required for its expression,
2 o 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.
3 0 To direct a zkun6 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 zkun6, or may be derived from
another secreted protein (e.g., t-PA) or synthesized d~ novo. The secretory
signal sequence is operably linked to the zkun6 DNA sequence, i.e., the two
sequences are joined in the correct reading frame and positioned to direct the
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22
newly sythesized polypeptide into the secretory pathway of the host cell.
Secretory signal sequences are corrunonly 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. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).
Cultured marrrunalian 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 Genefics 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. Patent No. 4,713,339; Hagen et al., U.S.
Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; and Ringoid,
U.S. Patent 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;
2 o 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 USA. In
general, strong transcription promoters are preferred, such as promoters from
SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288. Other
suitable promoters include those from metallothionein genes (U.S. Patent
Nos. 4,579,821 and 4,601,978) and the adenovirus major fate 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
3 o University Boulevard, Manassas, VA USA under accession numbers 98669
and 98668, respectively.
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
3 5 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
a
<,,,
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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
- 5 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.
to hygromycin resistance, multi-drug resistance, puromycin acetyltransferase)
can also be used.
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
15 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 Autographs califomica 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
2o 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 _ gerieral, Giick and Pastemak,
25 Molecular Biotechnology: Principles and Applications of Recombinant DNA,
ASM Press, Washington, D.C., 1994.
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
3 o for transforming S. cerevisiae cells with exogenous DNA and producing
recombinant poiypeptides therefrom are disclosed by, for example, Kawasaki,
U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373;
Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743;
and Murray et al., U.S. Patent No. 4,845,075. Transformed cells are selected
35 by phenotype determined by the selectable marker, commonly drug
resistance or the ability to grow in the absence of a particular nutrient
(e.g.,
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24
leucine). A preferred vector system for use in Saccharomyces cerevisiae is
the POT7 vector system disclosed by Kawasaki et al. (U.S. Patent 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.
Patent No. 4,599,311; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter,
U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S.
Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.
Transformation systems for other yeasts, including Hansenula polymorpha,
1 o Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,
Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and
Candida maltose are known in the art. See, for example, Gleeson et al., J.
Gen. Microbiol. 132:3459-3465, 1986 and Cregg, U.S. Patent No. 4,882,279.
Aspergillus cells may be utilized according to the methods of McKnight et al.,
U.S. Patent No. 4,935,349. Methods for transforming Acremonium
chrysogenum are disclosed by Sumino et al., U.S. Patent No. 5,162,228.
Methods for transforming Neurospora are disclosed by Lambowitz, U.S.
Patent No. 4,486,533. Production of recombinant proteins in Pichia
methanolica is disclosed in U.S. Patents No. 5,716,808, 5,736,383,
5,854,039, and 5,888,768.
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 zkun6 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
_30 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
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release the contents of the periplasmic space and recovering the protein,
thereby obviating the need for denaturation and refolding.
Transformed or transfected host cells are cultured according to
conventional procedures in a culture medium containing nutrients and other
5 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
to 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
15 25°C to 35°C. Liquid cultures are provided with sufficient
aeration by
conventional means, such as shaking of small flasks or sparging of
fermentors.
It is preferred to purify the proteins of the present invention to
X80% purity, more preferably to Z90% purity, even more preferably z95%
2 o 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.
25 Zkun6 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., BiolTechnol. _6: 1321-1325, 1988.
3 o Using methods known in the art, zkun6 proteins can be
produced glycosylated or non-glycosylated; pegylated or non-pegylated; and
may or may not include an initial methionine amino acid residue.
The zkun6 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 Vlia, factor IXa, factor Xa, factor Xla, factor
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26
Xlla, 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. Zkun6 proteins are also contemplated for use in
preservation of platelet function, organ preservation, and wound healing.
Zkun6 proteins may be useful in the treatment of conditions
to arising from an imbalance in hemostasis, including acquired coagulopathies,
primary fibrinolysis and fibrinolysis due to cirrhosis, and complications from
high-dose thromboiytic 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
2 o platelet dysfunction. In addition, patients with liver disease commonly
experience increased flbrinolysis due to an inability to maintain normal
levels
of ors-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
flbrinolysis 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
3o circulating level of ocz-antiplasmin (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. Ciin. Med 126:1377-1384, 1995).
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27
Additional antithrombotic uses of zkun6 proteins include
_ treatment or prevention of deep vein thrombosis, pulmonary embolism, and
post-surgical thrombosis.
Zkun6 proteins may also be used within methods for inhibiting
blood coagulation in mammals, such as in the treatment of disseminated
intravascular coagulation. Zkun6 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.
to 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.
Zkun6 proteins may also find therapeutic use in the blockage of
proteolytic tissue degradation. Proteolysis of extracelluiar matrix,
connective
tissue, and other tissues and organs is an element of many diseases. This
tissue destruction is beieived to be initiated when plasmin activates one or
more matrix metalloproteinases (e.g., collagenase and metallo-elastases).
Inhibition of plasrnin by zkun6 proteins may thus be beneficial in the
treatment
of these conditions.
2o 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
comial 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 rnay 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
3 0 11 fi: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
3 5 deformities and dysfunction (see, Amett, in Ceci! Textbook of Medicine,
ibid).
Osteoarthritis is a chronic disease causing deterioration of the joint
cartilage
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28
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 (Spir'tto, Inflam. Res. 44 (supp. 2):S131-S132, 1995; O'Byme,
lnflam. Res. 44 (supp. 2):S117-S118, 1995; Karran, Ann. Rheumatic Disease
54:662-669, 1995). Zkun6 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, hepatocefiular
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 coliagenous stromal tissue. Damage due to thermal or
chemical injury to corneal surfaces often results in a chronic wound-healing
s'ttuation. 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. Parhol. 149:1287-1302, 1996).
The zkun6 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 zkun6 protein may be
2o used in combination with tissue piasminogen activator in thromboiytic
therapy.
Doses of zkun6 proteins will vary according to the severity of the
condition being treated and may range from approximately 10 pg/kg to 10
mglkg body weight, preferably 100 p,g/kg to 5 mg/kg, more preferably 100
p,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 diiuent, and resuspended immediately prior to use.
3 0 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, Reminaton~ The Science and Practice of Pharmacy,
Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed., 1995.
Gene therapy provides an alternative therapeutic approach for
delivery of zkun6 proteins. If a mammal has a mutated or absent zkun6 gene,
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29
a polynucleotide encoding a zkun6 protein can be introduced into the cells of
the mammal. In one embodiment, a gene encoding a zkun6 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.
so 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. Viral. 63:3822-8, 1989).
Within another embodiment, a zkun6 polynucleotide can be
introduced in a retroviral vector, as described, for example, by Anderson et
al., U.S. Patent No. 5,399,346; Mann et al. Cell 33:153, 1983; Temin et al.,
U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289;
Markowitz et al., J. Virol. (2:1120, 1988; Temin et al., U.S. Patent 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. Aced Sci. USA 84:7413-7, 1987; Mackey et al.,
Proc. Natl. Acad Sci. USA 85:8027-31, 1988).
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
3 o 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, W a et al., J. Biol. Chem.
2 7:963-7, 1992; W a et al., J. Biol. Chem. 263:14621-4, 1988.
3 5 Zkunfi proteins can also be used to prepare antibodies that
specifically bind to zkun6 proteins. As used herein, the term "antibodies"
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includes polyclonal antibodies, monoclonal antibodies, antigen-binding
fragments thereof such as F(ab')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
5 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
1o 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
15 associated with particular antibody constant domains. Alternative
techniques
for generating or selecting antibodies useful herein include in vitro exposure
of lymphocytes to a zkun6 protein, and selection of antibody display libraries
in phage or similar vectors (for instance, through use of immobilized or
labeled zkun6 polypeptide). Antibodies are defined to be specifically binding
20 if they bind to a zkun6 protein with an affinity at least 10-fold greater
than the
binding affinity to control (non-zkun6) polypeptide. It is preferred that the
antibodies exhibit a binding affinity (Ka) of 106 M-' or greater, preferably
10'
M'' or greater, more preferably 108 M'' or greater, and most preferably 109
M''
or greater. The affinity of a monoclonal antibody can be readily detem~ined
25 by one of ordinary skill in the art (see, for example, Scatchard, Ann.
NYAcad.
Sci. 51: 660-672, 1949). .
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
3 o Raton, FL, 1982). As would be evident to one of ordinary skill in the art,
polyclonal antibodies can be generated from a variety of wamrblooded
animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice,
and rats. The immunogenicity of a zkun6 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 zkun6 protein or a portion
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3f
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.
Immunogenic zkun6 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 N0:2 includes
1 o residues 44 (Asn) - 54 (Asp).
A variety of assays known to those skilled in the art can be
utilized to detect antibodies that specifically bind to a zkun6 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.
Antibodies to zkun6 may be used for affinity purification of
2 o zkun6 proteins; within diagnostic assays for determining circulating
levels of
zkun6 proteins; for detecting or quantitating soluble zkun6 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.
Zkun6 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
CA 02342072 2003-09-08
32
during cell lysis. The proteins can also be used in the laboratory as a tissue
culture additive to prevent cell detachment.
The invention is further illustrated by the following non-limiting
example.
EXAMPLE ;w
To obtain a Zkun6 cDNA clone, cDNA is prepared from stomach
using a a commercially available kit (MarathonTM cDNA Amplification Kit from
Clontech Laboratories, Inc., Palo Alto, CA) and an oligo(dT) primer. To
amplify the zkun6 DNA, 5 ~.l each of 1/100 diluted cDNAs, 20 pmoles each of
two oligonucleotide primers designed from SEQ ID N0:1, and 1 U of a 2:i
mixture of ExTaqT'~ DNA ,polymerase (TaKaRa Biomsdicals) and Pfu ANA
polymerasse (Stratagene, La Jolla, CA) (ExTaqlPfu)* are used in a 25-~I
reaction mixture. The reaction mixture is incubated at 94°C for 2
minutes; 25
cycles of 94°C for 15 seconds, 66°C for 20 seconds, and
72°C for 30 seconds;
and a 1-minute incubation at 72°C. 1 p,l each of 11100 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-~,I
reaction mixtures. The mixtures are incubated at 94°C for 2 minutes; 2
cycles
of 94°C for 15 seconds, 66°C for 20 seconds, 72°C for 30
seconds; 25 cycles
of 94°C for 15 seconds, 64°C for 20 seconds, 72°C for 30
seconds; and a 1-
minute incubation at 72°C. The PCR products are gel purified and
sequenced
to confirm their identity.
To construct an expression vector for the zkun6 Kunitz domain,
2 5 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 subcioning into an expression vector. 5 p.l of
1/100 diluted cDNA, 20 pmoles of each oligonucleotide primer, and 1 U of
ExTaqIPfu are used in 25-~.I reaction mixtures. The mixtures are incubated at
94°C for 2 minutes; 3 cycles of 94°C for 30 seconds, 50°C
for 30 seconds,
72°C for 30 seconds; 35 cycles of 94°C for 30 seconds,
68°C for 30 seconds;
and a 7-minute incubation at 72°C. The PCR product is gel purified and
restriction digested with Bam HI and Xho I overnight.
3 5 The mammalian expression vector pZP-9 was constructed with
the dihyrofolate reductase gene selectable marker under control of the SV40
* trade-mark
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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. To facilitate protein purification, the pZP9 vector
was modified by addition of a tissue plasminogen activator (t-PA) secretory
signal sequence (see U.S. Patent No. 5,641,655) and a GIuGIu tag sequence --
(SEQ ID N0:6) 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
1o vector was designated pZP9NEE. The vector pZPNEE is digested with Bam
HI and Xho I, and the zkun6 fragment is inserted. The resulting construct is
confirmed by sequencing.
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.
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1
SEQUENCE LISTING
<110> ZymoGenetics, Inc.
<120> KUNITZ DOMAIN POLYPEPTIDE ZKUN6
<130> 98-40PC
<160> 7
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 177
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1)...(177)
<400> 1
ggc ccc ggc gac gcc tgc gtg ctg cct gcc gtg cag ggc ccc tgc cgg 48
Gly Pro Gly Asp Ala Cys Ual Leu Pro Ala Val Gln Gly Pro Cys Arg
1 5 10 15
ggc tgg gag ccg cgc tgg gcc tac agc ccg ctg ctg cag cag tgc cat 96
Gly Trp Glu Pro Arg Trp Ala Tyr Ser Pro Leu Leu Gln Gln Cys His
20 25 30
ccc ttc gtg tac ggt ggc tgc gag ggc aac ggc aac aac ttc cac agc 144
Pro Phe Ual Tyr Gly Gly Cys Glu Gly Asn Gly Asn Asn Phe His Ser
35 40 45
cgc gag agc tgc gag gat gcc tgc ccc gtg ccg 177
Arg Glu Ser Cys Glu Asp Ala Cys Pro Ual Pro
50 55
<210> 2
<211> 59
<212> PRT
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2
<213> Homo sapiens
<400> 2
Gly Pro Gly Asp Ala Cys Ual Leu Pro Ala Ual Gln Gly Pro Cys Arg
1 5 10 15
Gly Trp Glu Pro Arg Trp Ala Tyr Ser Pro Leu Leu Gln Gln Cys His --
20 25 30
Pro Phe Ual Tyr Gly Gly Cys Glu Gly Asn Gly Asn Asn Phe His Ser
35 40 45
Arg Glu Ser Cys Glu Asp Ala Cys Pro Ual Pro
50 55
<210> 3
<211> 51
<212> PRT
<213> Artificial Sequence
<220>
<223> Kunitz motif
<221> VARIANT
<222> (2)...(2)
<223> Xaa is any residue except Asp. Cys. Gly, His, Met.
Pro or Trp
<221> VARIANT
<222> (3)...(3)
<223> Xaa is Leu, Glu. Met, Gln, Phe, Ser, Thr, Ala or
Pro --
<221> VARIANT
<222> (4)...(4)
<223> Xaa is any residue except Arg, Cys, Met. Phe, Trp,
Tyr or Val
<221> VARIANT
<222> (5)...(5)
<223> Xaa is any residue except Asn, Cys, Gln, Gly, Phe,
w Ser, Thr or Trp
<221> VARIANT
<222> (6)...(6)
<223> Xaa is Arg, Glu, Asn, Ala. Val, Asp, Lys, Ser, Tyr
CA 02342072 2001-03-05
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3
or Met
<221> VARIANT
<222> (7)...(7)
<223> Xaa is any residue except Asn, Cys, Gly, His. Leu,
Met, Phe or Trp
<221> VARIANT
<222> (8)...(8)
<223> Xaa is Gly or Glu
<221> VARIANT
<222> (9)...(9)
<223> Xaa is Pro, Arg, Leu. Ual, Ser, Asp, Ile, Asn or
Thr
<221> VARIANT
<222> (11)...(11)
<223> Xaa is any residue except Ala. Cys, Glu. His. Ile,
Pro, Trp or Ual
<221> VARIANT
<222> (12)...(12)
<223> Xaa is Arg, Lys, Ala. Asp. Gln, Phe. Gly, Glu. Thr
or Ser
<221> VARIANT
<222> (13)...(13)
<223> Xaa is any residue except Asp, Cys~, Glu. Pro or Thr
<221> VARIANT
<222> (14)...(14)
<223> Xaa is any residue except Arg. Asn. Cys. Gly, His. Ser. Trp or
Tyr
<221> VARIANT
<222> (15)...(15)
' <223> Xaa is any residue except Ala. Asp, Cys, Gly, His, Met, Trp or
Tyr
<221> VARIANT
<222> (16)...(16)
<223> Xaa is Ser, Ala. Arg, Ual, Gln. Lys. Leu. G1y or Ile
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4
<221> VARIANT
<222> (17)...(17)
<223> Xaa is Phe, Tyr, Ile, Trp or Leu
<221> VARIANT
<222> (18)...(18)
<223> Xaa is Tyr, His, Phe. Trp, Asn or Ala
<221> VARIANT
<222> (19)...(19)
<223> Xaa is Tyr or Phe
<221> VARIANT
<222> (20)...(20)
<223> Xaa is Lys. Asn, Ser or Asp
<221> VARIANT
<222> (21)...(21)
<223> Xaa is any residue except Asp, Cys, Glu. His or Tyr
<221> VARIANT
<222> (22)...(22)
<223> Xaa is any residue except Cys, Met. Pro or Trp
<221> VARIANT
<222> (23)...(23)
<223> Xaa is Ala, Lys, Ser. Leu. Thr, Ile. Gln. Glu, Tyr or Val
<221> VARIANT
<222> (24)...(24)
<223> Xaa is Lys. Gln, Asn. His. Gly, Arg or Met
<221> VARIANT
<222> (25)...(25)
<223> Xaa is any residue except Asn, Asp, Cys, His. Ile, Pro, Trp,
Tyr or Ual
<221> VARIANT
<222> (27)...(27)
<223> Xaa is any residue except Cys, Gly, Phe, Pro. Ser or Trp
<221> VARIANT
<222> (28)...(28)
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<223> Xaa is any residue except Asp, Cys, His, Ile, Phe. Trp or Tyr
' <221> VARIANT
<222> (29)...(29)
<223> Xaa is Phe or Tyr
<221> VARIANT
<222> (30)...(30)
<223> Xaa is any residue except Arg, Cys, Gly or Met
<221> VARIANT
<222> (31)...(31)
<223> Xaa is Tyr, Trp, Phe or Asp
<221> VARIANT
<222> (32)...(32)
<223> Xaa is Ser, Gly or Thr
<221> VARIANT
<222> 133)...(33)
<223> Xaa is Gly or Ile
<221> VARIANT
<222> (35)...(35)
<223> Xaa is Gly. Lys, Arg. Pro, Gin, Leu, Glu, Asn or Met
<221> VARIANT
<222> (36)...(36)
<223> Xaa is Gly. Lys or Ala
<221> VARIANT
<222> (37)...(37)
<223> Xaa is Asn, Lys or Ser
<221> VARIANT
<222> (38)...(38)
' <223> Xaa is any residue except Cys. His, Ile, Phe, Pro, Thr. Trp,
Tyr or Val
<221> VARIANT
<222> (39)...(39)
<223> Xaa is Asn or Tyr
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6
<221> VARIANT
<222> (40)...(40)
.- <223> Xaa is Arg, Asn, Lys, Gln or Ual
<221> VARIANT
<222> (41)...(41)
<223> Xaa is Phe, Tyr or Asp
<221> VARIANT
<222> (42)...(42)
<223> Xaa is any residue except Cys. Gln, Gly, Phe or Trp
<221> VARIANT
<222> (43)...(43)
<223> Xaa is Thr. Ser. Arg, Lys or Asp
<221> VARIANT
<222> (44)...(44)
<223> Xaa is Ile, Leu, Trp, Arg, Lys, Thr, Glu, Ala, Gln or Val
<221> VARIANT
<222> (45)...(45)
<223> Xaa is Glu, Asp, Ala, His. Met, Ual, Gln, Lys, Arg or Pro
<221> VARIANT
<222> (46)...(46)
<223> Xaa is Glu, Lys, Gln, Asp, Ala, Tyr or Ser
<221> VARIANT
<222> (48)...(48)
<223> Xaa is any residue except Ala, Cys, Gly, Phe, Pro, Ser, Thr.
Trp or Tyr
<221> VARIANT
<222> (49)...(49)
<223> Xaa is any residue except Cys, Ile, Leu, Met, Phe, Pro, Ser,
Tyr or Val
'T <221> VARIANT
<222> (50)...(50)
<223> Xaa is Thr, Ala, Val, Ile. Phe, Leu, Met, Lys, Tyr or Arg
<400> 3
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7
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
<2I0> 4
<211> 177
<212> DNA
<213> Artificial Sequence
<220>
<223> Degenerate Sequence
<221> variation
<222> (1)...(177)
<223> n is any nucleotide
<400> 4
ggnccnggng aygcntgygt nytnccngcn gtncarggnc cntgymgngg ntgggarccn 60
mgntgggcnt aywsnccnyt nytncarcar tgycayccnt tygtntaygg nggntgygar 120
ggnaayggna ayaayttyca ywsnmgngar wsntgygarg aygcntgycc ngtnccn 177
<210> 5
<211> 55
<212> PRT
<213> Homo
sapiens
<400> 5
Thr Asp Cys LeuPro Lys Asp Gly Thr Arg Asp
Ile Lys Glu Cys Phe
1 5 10 15
Ile Leu Trp TyrAsp Pro Asn Lys Ser Ala Arg
Lys Tyr Thr Cys Phe
20 25 30
Trp Tyr Gly GlyGly Asn Glu Lys Phe Ser Gln
Gly Cys Asn Gly Lys
35 40 45
Glu Cys Lys CysAla
Glu Ual
50 55
<210> 6
<211> 6
CA 02342072 2001-03-05
WO 00/14235
PCT/E)S99/20202
8
<212> PRT
<213> Artificial Sequence
<220>
<223> Glu-Glu tag
<400> 6
Glu Tyr Met Pro Met Glu
1
<210> 7
<21I> 4
<212> PRT
<213> Artificial Sequence
<223> Thrombin cleavage site
<400> 7
Leu Ual Pro Arg
1
