WO2001066771A2 - Human serine protease - Google Patents

Abstract

Members of the serine protease family play a role in carefully controlled processes, such as blood coagulation, fibrinolysis, complement activation, fertilization, and hormone production. These enzymes are also used in a variety of diagnostic, therapeutic, and industrial contexts. Ztryp3 is a new member of the serine protease family.

Description

HUMAN SERINE PROTEASE

TECHNICAL FIELD

The present invention relates generally to a new gene that encodes an enzyme In particular, the present invention relates to a no\ el seπne protease, designated "Ztryp^ ^' and to nucleic acid molecules encoding Ztryp"

BACKGROUND OF THE I VENTION Endogenous proteolytic enzymes provide a

ot useful functions, including the degradation of inv ading organisms, antigen-antibodx complexes and certain tissue proteins that aie no longer necessan The seπne proteases comprise a large family of enzymes that use an activated seπne residue in the substrate-binding site to catalytically hydrolyze peptide bonds Typically, this seπne residue can be identified by the irreversible reaction of its side chain hydrox\ l group with dπsopropylfluorophosphate Seπne proteases paiticipate in carefulh controlled processes, such as blood coagulation, fibπnolysis. complement actu ation, fertilization. and hormone production These proteases are utilized in a vaπeπ of diagnostic and therapeutic contexts, and as industrial enzvmes Normally, seπne proteases catalyze limited proteoh si*.. in that only one or two specific peptide bonds of the protein substrate are cleaved Under denaturing conditions, seπne proteases can hydrolyze multiple peptide bond^ resulting in the digestion of peptides, proteins, and even autolysis Several diseases are thought to result from the lack of regulation of seπne protease activin . including emphysema arthritis, cancer metastasis, and thrombosis

For example, human tryptase is a seπne protease with trypsin-hke proteolytic activity (see, for example, Numerof. ct al E\p Opm hn est Dιu°s 6 81 1 ( 1997), Chan, et al , Pi ot Express Purif 75.251 ( 1999), Eli od and Numerof, Emeigms, Therapeutic Targets 3 203 ( 1999)) Tryptase is almost exclusu eh found in the secretory granules of mast cells, and the enzyme is released along with heparin and histamine upon mast cell activation in inflammatory response In humans, tryptase has been established as an important mediator of aπwa\ response and is implicated in increasing the magnitude of broncoconstπction in asthma In addition tryptase has been shown to stimulate cytokme production, and exhibit mitogemc effects in a vaπety of cell types, and therefore, the enzyme may also contribute to the hyperplasia and fibrotic changes observed in diseases such as asthma Animal model studies and clinical studies have shown that tryptase inhibitois are effective in reducing asthmatic response to exposure of antigen, and in blocking the mitogemc effects described abov e

Human tryptase has also been implicated in activities such as cleav ing fibπnogen α and β chains, collagen IV, gelatinase and fibronectin Accordingly the enzyme is implicated in the inhibition of coagulation and in tissue remodeling in the lung and other tissues Moreover, tivptase is show n to cleave calcitomn gene-related peptide

hich is a potent vasodilator, and hence tryptase

potentiate gastric ulceration or increase cutaneous neurogemc inflammation and promote smooth muscle conti action In addition tryptases are implicated in matπx degradation ound heanng and tumor metastasis

Therefore, the discovery of a new seπne protease fulfills a need in the art by providing a new composition useful in diagnosis therapv. or lndustiy

BRIEF SUMMARY OF THE INVENTION The present invention provides a novel serine protease designated

"Ztryp3 ^*' The present invention also provides Ztryp3 variant polypeptides and Ztrvp3 fusion proteins, as well as nucleic acid molecules encoding such polypeptides and proteins, and methods for using these nucleic acid molecules and ammo acid sequences

DETAILED DESCRIPTION OF THE INVENTION

1. Overview

The present invention provides nucleic acid molecules that encode a new human serine protease, designated as "Ztryp3 " An illustrative nucleotide sequence that encodes Ztryp3 is provided by SEQ ID NO 1 The encoded polypeptide has the following amino acid sequence MKYVFYLGVL AGTFFFADSS VQKEDPAPYL

VYLKSHFNPC VGVLIKPSWV LAPAHCYLPN LKVMLGNFKS RVRDGTEQTI

NPIQIVRYWN YSHSAPQDDL MLIKLAKPAM LNPKVQPLTL ATTNVRPGTV CLLSGLDWSQ ENSGRHPDLR QNLEAPVMSD RECQKTEQGK SHRNSLCVKF

VKVFSRΓFGE VAVATVICKD KLQGBΞVGHF MGGDVGIYTN VYKYVSWIEN TAKDK (SEQ ID NO 2) Thus, the Ztr p3 gene described herein encodes a polypeptide of 235 amino acids, as shown in SEQ ID NO.2

The Ztr\p3 gene is expressed in testicular tissue mammary gland, salivary gland, and spinal cord In contrast, little or no Ztr\p3 gene expression was detected in adrenal gland, lung, bladder, colon, liver, pituitarv , placenta, uterus, and prostate tissues Moreover. Ztnp3 gene expression was detected normal ovarian tissue, but not in ovarian tumor tissue These results indicate that Ztι \p3 nucleotide sequences and antι-Ztryp3 antibodies can be useful for tissue differentiation

Ztryp3 has an unglycosylated moleculai weight of 26.443 Daltons Sequence analysis indicates that the Ztryp3 signal sequence resides in amino acid residues 1 to 19 of SEQ ID NO 2 Analysis of the Ztryp3 sequence also revealed that two ammo acid residues of the seπne protease catalytic triad are provided by His^" and Asp"' The third membei of the active site can be provided b\ Sei^9"" or Ser^g" According to structural analysis. Ztryp3 can contain at least one intra-chain disulfide bond Ztryp3 may contain as many as three intra-chain disulfide bonds at Cys⁴⁰ - Cys^% Cys^{π ι} - Cys¹⁹⁸, and Cys¹⁶¹ - Cys'⁷⁷

As detailed below , the present invention provides isolated polypeptides having an amino acid sequence that is at least 70%. at least 809c, or at least 90% identical to the amino acid sequence of SEQ ID NO.2, or to amino acid residues 20 to 235 of SEQ ID NO.2 Particular polypeptides specifically bind with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO.2 Particular polypeptides also can be characterized by serine protease activity

An illustrative polypeptide is a polypeptide that comprises the amino acid sequence of SEQ ID NO.2. or that comprises ammo acid residues 20 to 235 of SEQ ID NO.2 Additional exemplary polypeptides include polypeptides comprising an amino acid sequence of 15, 20. or 30 contiguous amino acids of an amino acid sequence selected from the group consisting of amino acid residues 20 to 235 of SEQ ID NO.2, amino acid residues 20 to 130 of SEQ ID NO.2, amino acid residues 20 to 198 of SEQ ID NO.2, amino acid residues 40 to 198 of SEQ ID NO 2. and SEQ ID NO.2 Additional examples of a Ztryp3 polypeptide include polypeptides consisting of, or comprising, any of the following amino acid sequences amino acid iesidues 20 to 235 of SEQ ID NO.2. amino acid residues 20 to 130 of SEQ ID NO.2, amino acid residues 20 to 198 of SEQ ID NO.2. and amino acid residues 40 to 198 of SEQ ID NO.2 Nucleic acid molecule encoding these amino acid sequences aie useful as probes and to produce the encoded polypeptides

The present invention further provides antibodies and antibody fragments that specifically bind with such polypeptides Exemplar, antibodies include polyclonal antibodies, muπne monoclonal antibodies, humanized antibodies derived from muπne monoclonal antibodies, and human monoclonal antibodies Illustrativ e antibody fragments include F(ab') , F(ab)₂. Fab . Fab. Fv. scFv, and minimal recognition units The present invention further includes compositions comprising a carrier and a peptide, polypeptide, antibody, or anti-idiotype antibody described herein

The present invention also provides isolated nucleic acid molecules that encode a Ztryp3 polypeptide. wherein the nucleic acid molecule is selected from the group consisting of a nucleic acid molecule having the nucleotide sequence of SEQ ID NO 3. a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO 2 and a nucleic acid molecule that remains hybridized following stringent wash

to a nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of (a) the nucleotide sequence of SEQ ID NO 1. (b) nucleotides 42 to 746 of SEQ ID NO 1 (c) nucleotides 99 to 746 of SEQ ID NO 1 , and (d) a nucleotide sequence that is the complement of the nucleotide sequence of (a), (b), or (o Illustrative nucleic acid molecules include those in which anv difference between the amino acid sequence encoded by the nucleic acid molecule and the corresponding amino acid sequence of SEQ ID NO 2 is due to a conservative amino acid substitution The present invention furthei contemplates isolated nucleic acid molecules that comprise the nucleotide sequence of SEQ ID NO 1 , or nucleotides 99 to 746 of SEQ ID NO.1

The present invention also includes vectors and expression vectors comprising such nucleic acid molecules Such expression vectois ma\ comprise a transcription promoter, and a transcription terminator wherein the promoter is operabh linked with the nucleic acid molecule, and wherein the nucleic acid molecule is operably linked with the transcription terminator The present invention further includes recombinant host cells comprising these vectois and expression vectors Illustrative host cells include bacterial, yeast, fungal, insect, mammalian, and plant cells Recombinant host cells comprising such expression vectors can be used to produce Ztryp3 polypeptides by cultuπng such recombinant host cells that comprise the expression vector and that produce the Ztryp3 protein, and, optionally, isolating the Ztryp3 protein from the cultured recombinant host cells The present inv ention also includes the piotein products of these processes

The present invention also contemplates methods for detecting the presence of Ztrvp 3 RNA in a biological sample, comprising the steps of (a) contacting a Ztryp3 nucleic acid probe under hybridizing conditions with either (I) test RNA molecules isolated from the biological sample, or (n) nucleic acid molecules synthesized from the isolated RNA molecules wherein the probe has a nucleotide sequence comprising a portion of the nucleotide sequence of SEQ ID NO 1. or its complement, and (b) detecting the formation of hybrids of the nucleic acid probe and either the test RNA molecules or the synthesized nucleic acid molecules wherein the presence of the hybrids indicates the presence of Ztr\p3 RNA in the biological sample An example of a biological sample is a human biological sample such as a biopsy or autopsy specimen

The present invention further provides methods for detecting the presence of Ztryp3 polypeptide in a biological sample, comprising the steps of (a) contacting the biological sample with an antibody or an antibody fragment that specifically binds with a polypeptide comprising the amino acid sequence of amino acid residues 20 to 235 of SEQ ID NO 2, wherein the contacting is performed under conditions that allow the binding of the antibody or antibodv fragment to the biological sample, and (b) detecting any of the bound antibody or bound antibody fragment Such an antibody or antibody fragment may further comprise a detectable label selected from the group consisting of radioisotope. fluorescent label, chemiluminescent label, enzyme label, bioluminescent label, and colloidal gold An exemplary biological sample is a human biological sample

The present invention also provides kits for performing these detection methods For example, a kit for detection of Ztr\p3 gene expression may comprise a container that comprises a nucleic acid molecule, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO 1 , (b) a nucleic acid molecule comprising the complement of the nucleotide sequence of SEQ ID NO 1 , (c) a nucleic acid molecule that is a fragment of (a) consisting of at least eight nucleotides. and (d) a nucleic acid molecule that is a fragment of (b) consisting of at least eight nucleotides Illustrative nucleic acid molecules include nucleic acid molecules comprising nucleotides 99 to 746 of SEQ ID NO 1, or the complement thereof Such a kit may also comprise a second container that comprises one or more reagents capable of indicating the presence of the nucleic acid molecule On the other hand, a kit for detection of Ztryp3 protein ma comprise a container that comprises an antibodv. or an antibody fragment, that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO 2

The present invention further provides fusion proteins a Ztryp3 polypeptide and an immunoglobuhn moiety In such fusion proteins, the immunoglobulin moiety may be an immunoglobuhn heavy chain constant region, such as a human F_c fragment The present invention further includes isolated nucleic acid molecules that encode such fusion proteins These and other aspects of the inv ention w ill become evident upon reference to the following detailed description In addition v arious refeiences aie identified below

^ 2. Definitions

In the description that follow s, a number of terms are used extensiv e!} The following definitions are provided to facilitate understanding of the invention

As used herein, "nucleic acid 01 nucleic acid molecule refeis to l o poly nucleotides such as deoxyπbonucleic acid (DN A) or πbonucleic acid (RN A. ) oligonucleotides fragments generated bv the polvmerase chain reaction (PCR ) and fragments generated by anv of hgation, scission, endonuclease action and εxonuclease action Nucleic acid molecules can be composed of monomei s that are naturalh occurring nucleotides (such as DNA and RNA). oi analogs of naturally-occurring

1 nucleotides (e g α-enantiomeπc forms of natuiallv -occurring nucleotides) or a combination of both Modified nucleotides can hav e alterations in sugar moieties and/or in pyπmidine or purme base moieties Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido gioups, or sugai s can be functiona zed as ethers or esters 0 Moreovei, the entne sugar moιet> can be replaced w ith stericallv and electronicall) similar structures, such as aza-sugars and caibocyc c sugar analogs Examples of modifications in a base moiety include alkylated puπnes and pyπmidines, acylated puπnes or pyπmidines. or other well-know n heterocvchc substitutes Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages Analogs 5 of phosphodiester linkages include phosphorothioate. phosphorodithioate. phosphoroselenoate, phosphorodiselenoate, phosphoioanilothioate, phosphoramhdate, phosphoramidate, and the like The term "nucleic acid molecule^" also includes so- called ' peptide nucleic acids.^" which comprise natural! v-occurπng or modified nucleic acid bases attached to a polyamide backbone Nucleic acids can be either single 0 stranded or double stranded

The term "complement of a nucleic acid molecule^* refers to a nucleic acid molecule having a complementary nucleotide sequence and reverse orientation as compared to a reference nucleotide sequence Foi example the sequence 5 ATGCACGGG 3 is complementary to 5' CCCGTGCAT 3 5 The term "contig" denotes a nucleic acid molecule that has a contiguous stretch of identical or complementary sequence to another nucleic acid molecule Contiguous sequences are said to ' overlap^' a given stretch of a nucleic acid molecule either in their entirety or along a partial stretch of the nucleic acid molecule

The term "degenerate nucleotide sequence" denotes a sequence of nucleotides that includes one or more degenerate codons as compared to a reference nucleic acid molecule that encodes a polypeptide Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i e GAL and GAC triplets each encode Asp)

The term "structural gene^" refers to a nucleic acid molecule that is tianscπbed into messenger RNA (mRNA). which is then translated into a sequence of amino acids characteristic of a specific polypeptide

An ' isolated nucleic acid molecule^' is a nucleic acid molecule that is not integrated in the genomic DNA of an organism Foi example, a DNA molecule that encodes a grow th factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule Another example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule that is not integrated in the genome of an organism A nucleic acid molecule that has been isolated from a particular species is smaller than the complete DNA molecule of a chromosome from that species

A "nucleic acid molecule construct" is a nucleic acid molecule, either single- or double-stranded, that has been modified through human intervention to contain segments of nucleic acid combined and juxtaposed in an arrangement not existing in nature

"Linear DNA" denotes non-circular DNA molecules having fiee 5 and 3' ends Linear DNA can be prepared from closed circular DNA molecules, such as plasmids, by enzymatic digestion or physical disruption "Complementary DNA (cDNA)^' is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme rev erse transcπptase Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcπption Those skilled in the art also use the term "cDNA" to refer to a double- stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand The term "cDNA" also refers to a clone of a cDNA molecule synthesized from an RNA template

A "promoter" is a nucleotide sequence that directs the transcription of a structural gene Typically, a promoter is located in the 5 non-coding region of a gene proximal to the transcπptional start site of a structural gene Sequence elements w ithin promoters that function in the initiation of tianscπption are often characterized b> consensus nucleotide sequences These promoter elements include RNA polymerase binding sites, TATA sequences CAAT sequences, differentiation-specific elements (DSEs, McGehee et al . Mol Endocrinol 7 551 (1993)), cyclic AMP response elements (CREs). serum response elements (SREs, Treisman. Seminais in Cancel Biol 1 47 (1990)), glucocorticoid response elements (GREs), and binding sites for other transcription factors, such as CRE/ATF (O Reilly et al J Biol Chem 267 19938 - (1992)), AP2 (Ye et al . J Biol Chem 269 25728 (1994)) SP1. cAMP response element binding protein (CREB, Loeken, Gene

3 253 ( 1993)) and octamer factors (see in general. Watson et al eds , Moleculai Bιolog\ of the Gene, 4th ed (The Beniamin/Cummings Publishing Company, Inc 1987), and Lemaigre and Rousseau, Biochem J 303 1 ( 1994)) If a promoter is an inducible promoter, then the rate of

K transcription increases in response to an inducing agent In contrast the rate of transcription is not regulated bv an inducing agent if the promoter is a constitutive piomotei Repressible promoters are also known

A ' core promoter contains essential nucleotide sequences for promoter function, including the TATA box and start of transcription Bv this definition, a core

1 promoter may or may not have detectable activity in the absence of specific sequences that may enhance the activity or confer tissue specific activity

A "regulatory element" is a nucleotide sequence that modulates the activity of a core promoter For example, a regulatory element may contain a nucleotide sequence that binds with cellular factois enabling transcription exclusively 0 or preferentially in particular cells, tissues, or organelles These types of regulatory elements are normally associated with genes that are expiessed in a ' cell-specific." "tissue-specific." or "organelle-specific^" manner

An "enhancer" is a type of regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer 5 relative to the start site of transcription

"Heterologous DNA" lefers to a DNA molecule, or a population of DNA molecules, that does not exist naturally within a given host cell DNA molecules heterologous to a particular host cell may contain DNA derived from the host cell species (i e endogenous DNA) so long as that host DNA is combined with non-host 0 DNA (i.e , exogenous DNA) For example, a DNA molecule containing a non-host DNA segment encoding a polypeptide operably linked to a host DNA segment comprising a transcription promoter is considered to be a heterologous DNA molecule Conversely, a heterologous DNA molecule can comprise an endogenous gene operably linked with an exogenous promoter As another illustration, a DNA molecule 5 comprising a gene derived from a wild-type cell is considered to be heterologous DNA if that DNA molecule is introduced into a mutant cell that lacks the wild-type gene A "polypeptide^" is a polymer of amino acid residues joined by peptide bonds, whether pioduced naturally or synthetically Polypeptides of less than about 10 amino acid lesidues are commonly referred to as "peptides "

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 v ary w ith the type of cell Proteins are defined herein in terms of their amino acid backbone structures, substituents such as carbohydrate groups aie generally not specified, but mav be piesent nonetheless

A peptide or polypeptide encoded by a non-host DNA molecule is a "heterologous peptide or polypeptide

An "integrated genetic element^' is a segment of DNA tnat h been incorporated into a chromosome of a host cell after that element is introduced into the cell through human manipulation Within the piesent invention, integrated genetic elements are most commonly derived from linearized plasmids that are introduced into the cells by electroporation or other techniques Integrated genetic elements are passed from the original host cell to its progeny

A "cloning vector" is a nucleic acid molecule, such as a plasmid, cosrmd, or bacteπophage. that has the capability of replicating autonomously in a host cell Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites that allow insertion of a nucleic acid molecule in a determmable fashion without loss of an essential biological function of the vector, as well as nucleotide sequences encoding a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector Marker genes typically include genes that provide tetracyc ne resistance or ampicilhn resistance

An "expression vector^" is a nucleic acid molecule encoding a gene that is expressed in a host cell Typically, an expression vector comprises a transcription promoter, a gene, and a transcription terminator Gene expression is usually placed under the control of a promoter, and such a gene is said to be ' operably linked to" the promoter Similarly, a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter

A "recombinant host" is a cell that contains a heteiologous nucleic acid molecule, such as a cloning vector or expiession vector In the present context, an example of a recombinant host is a cell that produces Ztryp3 from an expression vector In contrast, Ztryp3 can be produced by a cell that is a ' natural source" of Ztryp3 (e g testis tissue), and that lacks an expression v ectoi "Integrative transformants" are recombinant host cells, in which heterologous DNA has become integrated into the genomic DNA of the cells

A "fusion protein^" is a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least t o genes. For example, a fusion protein can comprise at least part of a Ztryp3 polypeptide fused with a polypeptide that binds an affinity matrix. Such a fusion protein provides a means to isolate large quantities of Ztryp3 using affinity chromatography.

The term "receptor" denotes a cell-associated protein that binds to a bioactive molecule termed a "ligand ^" This interaction mediates the effect of the hgand on the cell. Receptors can be membrane bound, cytosohc or nuclear, monomeπc (e.g , thyroid stimulating hormone receptor, beta-adrenergic receptor) oi multimeπc (e.g , PDGF receptor, growth hormone receptor. IL-3 receptoi. GM-CSF receptor. G-CSF receptoi. erythropoietm receptor and IL-6 receptor) Membrane-bound receptors are characterized by a multi-domain structuie comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. In certain membrane-bound receptors, the extracellular ligand-binding domain and the intracellular effector domain are located in separate polypeptides that comprise the complete functional receptor.

In general, the binding of ligand to receptor results in a conformational change in the receptor that causes an intei action between the effector domain and other molecule(s) in the cell, which in turn leads to an alteration in the metabolism of the cell. Metabolic events that are often linked to receptor-hgand interactions include gene transcription, phosphorylation. dephosphorylation. increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane pids. cell adhesion, hydrolysis of inositol hpids and hydrolysis of phosphohpids.

The term "secretory signal sequence" denotes a nucleotide sequence that encodes a peptide (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.

An "isolated polypeptide" is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid. or other proteinaceous impurities associated with the polypeptide in nature. Typically, a preparation of isolated polypeptide contains the polypeptide in a highly purified form. i.e.. at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure. One way to show that a particular protein preparation contains an isolated polypeptide is by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamιde gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining of the gel However, the term "isolated^" does not exclude the presence of the same polypeptide in alternativ e physical forms, such as dimers or alternatively glycosylated or deπvatized forms 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 withm a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carbox l terminus of the complete polypeptide

The term "expiession^" refers to the biosyntnesis of a gene product Foi example, in the case of a stiuctural gene, expression inv olv es transcription ot the structural gene into mRNA and the translation of mRNA into one or more polypeptides 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 polypeptide encoded by a splice variant ot an mRNA transcribed from a gene

As used herein, the term "immunomodulator^" includes cytokines. stem cell growth factors, lymphotoxins. co-stimulatory molecules, hematopoietic factors, and synthetic analogs of these molecules The term "complement/anti-complement pair" denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair Othei exemplary complement/anti-complement pairs include receptor/hgand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like Wheie subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of less than 10⁹ M '

An "anti-idiotype antibody " is an antibody that binds with the variable region domain of an immunoglobuhn In the present context, an anti-idiotype antibody binds with the variable region of an antι-Ztryp3 antibody , and thus, an anti-idiotype antibody mimics an epitope of Ztryp3 Particular Ztryp3 anti-idiotype antibodies possess serine protease activity An ' antibody fragment' is a portion of an antibody such as F( ab )-> F(ab)₂, Fab . Fab. and the like Regardless of structure, an antibody fragment binds w ith the same antigen that is recognized by the intact antibody For example, an antι-Ztryp3 monoclonal antibodv fragment binds with an epitope of Ztryp3

The term ' antibody fragment" also includes a synthetic or a genetically engineered polypeptide that binds to a specific antigen, such as polypeptides consisting of the light chain variable region, "Fv " fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (' scFv proteins^' ) and minimal recognition units consisting of the amino acid residues that mimic the hypei variable iegion

A "chimeπc antibody^' is a iecombinant protein that contains the v ariable domains and complementary determining regions derived fiom a rodent antibodv hile the remainder of the antibody molecule is derived fiom a human antibody "Humanized antibodies" are recombinant proteins in which muπne complementarity determining legions of a monoclonal antibody have been transferred from heavy and light variable chains of the muπne immunoglobuhn into a human variable domain

As used herein, a "therapeutic agent" is a molecule or atom which is conjugated to an antibody moiety to produce a conjugate which is useful for therapy Examples of therapeutic agents include drugs, toxins, immunomodulators chelators, boron compounds, photoactive agents or dyes, and ladioisotopes

A "detectable label^" is a molecule or atom which can be conjugated to an antibody moiety to produce a molecule useful for diagnosis Examples of detectable labels include chelators, photoactive agents radioisotopes, fluorescent agents, paramagnetic ions, or other marker moieties

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 othei specific binding agent is available can be used as an affinity tag Affinity tags include a poly- histidme tract, protein A (Nilsson et al , EMBO J 4 1075 (1985), Nilsson et al Methods Enz\mol 198 3 (1991 )), glutathione S transferase (Smith and Johnson, Gene 67 31 (1988)), Glu-Glu affinity tag (Grussenmeyer et al Proc Natl Acad Sci USA 82 7952 (1985)). substance P, FLAG peptide (Hopp et al ,

6 1204 (1988)). streptavidin binding peptide. or other antigenic epitope or binding domain See. in general. Ford et al Protein Expression and Pui ificatwn 2 95 (1991 ) Nucleic acid molecules encoding affinity tags aie available from commercial suppliers (e g Pharmacia Biotech, Piscataway, NJ)

A ' naked antibody^" is an entire antibody as opposed to an antibody fragment, which is not conjugated with a therapeutic agent Naked antibodies include both polyclonal and monoclonal antibodies, as well as certain recombinant antibodies such as cfnmeπc and humanized antibodies

As used herein, the term "antibody component includes both an entire antibody and an antibody fragment

An "immunoconjugate" is a conjugate of an antibodv component with a therapeutic agent or a detectable label

As used herein the term antibodv fusion protein refeis to a recombinant molecule that comprises an antibodv component and a therapeutic agent

Examples of therapeutic agents suitable foi such fusion proteins include immunomodulators ( antibody-immunomodulator fusion protein^' ) and toxins ("antibody-toxin fusion protein' )

A "target polypeptide" or a "target peptide is an amino acid sequence that comprises at least one epitope, and that is expressed on a target cell, such as a tumor cell, or a cell that carries an infectious agent antigen T cells recognize peptide epitopes presented by a major histocompatibihty complex molecule to a target polypeptide or target peptide and typically lyse the target cell or recruit other immune cells to the site of the target cell, thereby killing the target cell

An "antigemc peptide" is a peptide, w hich will bind a major histocompatibihty complex molecule to form an MHC-peptide complex, which is recognized by a T cell thereby inducing a cytotoxic lvmphocyte response upon presentation to the T cell Thus, antigemc peptides are capable of binding to an appropriate major histocompatibihty complex molecule and inducing a cytotoxic T cells response, such as cell lysis or specific cytokine release against the target cell, which binds or expresses the antigen The antigemc peptide can be bound in the context of a class I or class II major histocompatibihty complex molecule, on an antigen presenting cell or on a target cell

In eukaryotes, RNA polymerase II catalyzes the transcription of a structural gene to produce mRNA A nucleic acid molecule can be designed to contain an RNA polymerase II template in which the RNA transcript has a sequence that is complementary to that of a specific mRNA The RNA transcript is teimed an ' anti- sense RNA" and a nucleic acid molecule that encodes the anti-sense RNA is termed an "anti-sense gene " Anti-sense RNA molecules are capable of binding to mRNA molecules, resulting in an inhibition of mRNA translation An "anti-sense ohgonucleotide specific for Ztryp3^" or an "Ztryp3 anti- sense oligonucleotide" is an ohgonucleotide having a sequence (a) capable of forming a stable triplex with a portion of the Ztr\p3 gene, or (b) capable of forming a stable duplex with a portion of an mRNA transcript of the Ztι \p3 gene A "πbozyme" is a nucleic acid molecule that contains a catalytic center

The term includes RNA enzymes, self-splicmg RNAs, self-cleaving RNAs. and nucleic acid molecules that perform these catalytic functions A nucleic acid molecule that encodes a πbozyme is termed a "πbozyme gene "

An "external guide sequence" is a nucleic acid molecule that directs the endogenous πbozyme, RNase P. to a particular species of intracellular mRNA, resulting in the cleavage of the mRNA by RNase P A nucleic acid molecule that encodes an external guide sequence is termed an "external guide sequence gene ^'

The term ' variant Ztnp3 gene refers to nucleic acid molecule^ that encode a polypeptide having an amino acid sequence that is a modification of SEQ ID NO 2 Such variants include naturally-occurring polymorphisms of Ztnp3 genes, as well as synthetic genes that contain conservative amino acid substitutions of the amino acid sequence of SEQ ID NO.2 Additional variant forms of Ztr\p3 genes are nucleic acid molecules that contain insertions or deletions of the nucleotide sequences described herein A variant

gene can be identified by determining whether the gene hybridizes with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO 1. or its complement, under stringent conditions

Alternatively, variant Ztr\p3 genes can be identified by sequence comparison Two amino acid sequences have "100%- amino acid sequence identity^" if the amino acid residues of the two amino acid sequences are the same when aligned tor maximal correspondence Similarly, two nucleotide sequences have "100% nucleotide sequence identity" if the nucleotide residues of the two nucleotide sequences are the same when aligned for maximal correspondence Sequence comparisons can be performed using standard software programs such as those included in the LASERGENE bioinformatics computing suite, which is produced by DNASTAR (Madison. Wisconsin) Other methods for comparing two nucleotide or amino acid sequences by determining optimal alignment are well-known to those of skill in the art (see. for example, Peruski and Peruski. The Internet and the New Biologλ Tools foi Genomic and Molecular Research (ASM Press, Inc 1997). Wu et al (eds ). "Information Superhighway and Computer Databases of Nucleic Acids and Proteins.^" in Methods in Gene Bιotechnolog\, pages 123-151 (CRC Press, Inc 1997). and Bishop (ed.). Guide to Human Genome Computing, 2nd Edition (Academic Press. Inc 1998)) Particular methods for determining sequence identity are described below Regardless of the particulai method used to identify a variant Ztr\p3 gene 01 variant Ztryp3 polypeptide. a variant gene or polypeptide encoded by a variant gene may be characterized by at least one of: the ability to bind specifically to an anti- Ztryp3 antibody, and seπne protease activity The term "allelic v ariant^" is used herein to denote any of two or moie 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 v ariant is also used herein to denote a protein encoded by an allelic variant of a gene

The term "oitholog^" 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 lesult of speciation

"Paralogs^" are distinct but structurally related proteins made by an organism Paralogs are believed to arise through gene duplication For example, α- globin, β-globm, and myoglobin are paralogs of each other

The present invention includes functional fragments of Ztnp3 genes. Within the context of this invention, a "functional fragment" of a Ztr\p3 gene refers to a nucleic acid molecule that encodes a portion of a Ztryp3 polypeptide which specifically binds with an antι-Ztryp3 antibody or possesses serine protease activity For example, a functional fragment of a Ztryp3 gene described herein comprises a portion of the nucleotide sequence of SEQ ID NO 1 , and encodes a polypeptide that specifically binds with an antι-Ztryp3 antibody

Due to the impiecision of standard analytical methods, molecular weights and lengths of polymers are 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%

3. Production of Nucleic Acid Molecules Encoding Ztryp3 Nucleic acid molecules encoding a human Ztnp3 gene can be obtained by screening a human cDNA or genomic library using polynucleotide probes based upon SEQ ID NO: 1 These techniques are standard and well-established

As an illustration, a nucleic acid molecule that encodes a human Ztryp3 can be isolated from a human cDNA libraiy In this case, the first step would be to prepare the cDNA library by isolating RNA from tissue (e.g , testicular tissue), using methods well-known to those of skill in the art In general. RNA isolation techniques must provide a method for breaking cells, a means of inhibiting RNase-directed degradation of RNA, and a method of separating RNA from DNA, protein, and polysacchaπde contaminants. For example, total RNA can be isolated by freezing tissue in liquid nitrogen, grinding the frozen tissue with a mortar and pestle to lyse the cells. extracting the ground tissue with a solution of phenol/chloroform to remove proteins, and separating RNA from the remaining impuπties by selective precipitation with lithium chloride (see, for example. Ausubel et al (eds ), Short Pioiocols in Moleculai Bwlogx 3"' Edition, pages 4-1 to 4-6 (John Wiley & Sons 1995) ["Ausubel (1995)"] Wu et al . Methods in Gene Biotechnologx , pages 33-41 (CRC Press. Inc 1997) ["Wu (1997)"] ) Alternatively, total RNA can be isolated from tissue by extracting giound tissue with guamdimum isothiocyanate, extracting with organic solvents, and sepaiating RNA from contaminants using differential centπfugation (see. for example. Chirgw in et al Biochemist) \ 18.52 ( 1979). Ausubel ( 1995 ) at pages 4-1 to 4-6. Wu ( 1997) at pages 33-41 ) In order to construct a cDNA library , poly(A)^{^} RNA must be isolated from a total RNA preparation Poly(A)⁺ RNA can be isolated from total RNA using the standard technique of ohgo(dT)-cellulose chromatography (see, for example, Aviv and Leder, Proc. Nat'l Acad. Sci. USA 69- 1408 (1972), Ausubel (1995) at pages 4-1 1 to 4- 12) Double-stranded cDNA molecules aie synthesized from poly(A)⁺ RNA using techniques well-known to those in the art (see. for example. W u (1997) at pages 41 -46) Moreover, commercially available kits can be used to synthesize double- stranded cDNA molecules For example, such kits are av ailable from Life Technologies. Inc (Gaithersburg. MD). CLONTECH Laboratories. Inc (Palo Alto. CA), Promega Corporation (Madison, WI) and STRATAGENE (La Jolla, CA)

Various cloning vectors are appropriate for the construction of a cDNA library For example, a cDNA library can be prepared in a vector derived from bacteπophage, such as a λgtlO vector See, for example. Huynh et al., "Constructing and Screening cDNA Libraries in λgtlO and λgtl l ," in DNA Cloning- A Piactical Approach Vol. 1. Glover (ed.), page 49 (TRL Press. 1985). Wu (1997) at pages 47-52

Alternatively, double-stranded cDNA molecules can be inserted into a plasmid vector, such as a PBLUESCRIPT vector (STRATAGENE. La Jolla. CA). a LAMDAGEM-4 (Promega Corp ) or other commercially available vectors Suitable cloning vectors also can be obtained from the American Type Cultuie Collection (Manassas, VA)

To amplify the cloned cDNA molecules, the cDNA library is inserted into a prokaryotic host, using standard techniques For example, a cDNA library can be introduced into competent E coh DH5 cells, which can be obtained, for example, from

Life Technologies, Inc. (Gaithersburg, MD)

A human genomic library can be prepared by means well-known in the art

(see. for example. Ausubel (1995) at pages 5-1 to 5-6: Wu (1997) at pages 307-327) - Genomic DNA can be isolated by lysing tissue with the detergent Sarkosyl. digesting the lysate with proteinase K, clearing insoluble debris from the lysate by centπfugation, precipitating nucleic acid from the lysate using isopropanol. and purifying resuspended

DNA on a cesium chloride density gradient

DNA fragments that are suitable for the production of a genomic library 10 can be obtained by the random shearing of genomic DNA or by the partial digestion of genomic DNA with restriction endonucleases Genomic DNA fragments can be inserted into a vector, such as a bacteπophage or cosmid vector, in accordance with conventional techniques, such as the use of restriction enzyme digestion to prov iαe appropriate termini. the use of alkaline phosphatase treatment to avoid undesirable joining of DN A molecules. I s and hgation with appropriate ligases Techniques for such manipulation are well-known in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6; W u (1997) at pages 307-

327)

Nucleic acid molecules that encode a human

gene can also be obtained using the polymerase chain reaction (PCR) with ohgonucleotide primers 0 having nucleotide sequences that are based upon the nucleotide sequences of the human

Ztr\p3 gene, as descπbed herein General methods for screening libraries w ith PCR are provided by. for example, Yu et al. "Use of the Polymerase Chain Reaction to Screen

Phage Libraries.^" in Methods in Molecular Bwlogλ Vol 15 PCR Protocols Current

Methods and Applications, White (ed ). pages 21 1-215 (Humana Press. Inc 1993) 5 Moreovei. techniques for using PCR to isolate related genes are described by. for example, Preston, "Use of Degenerate Ohgonucleotide Pπmeis and the Polymerase

Chain Reaction to Clone Gene Family Members." in Methods Molecular Biologx,

Vol. 15: PCR Protocols Current Methods and Applications, White (ed ) pages 317-

337 (Humana Press. Inc 1993) 0 Alternatively, human genomic libraries can be obtained from commercial sources such as Research Genetics (Huntsville, AL) and the American Type Culture

Collection (Manassas, VA)

A library containing cDNA or genomic clones can be screened with one or more polynucleotide probes based upon SΕQ ID NO: l . using standard methods (see. for 5 example. Ausubel (1995) at pages 6-1 to 6-11)

Antι-Ztryp3 antibodies, produced as descπbed below, can also be used to isolate DNA sequences that encode human Ztr\p3 genes from cDNA libraries For example, the antibodies can be used to screen λgtl l expression libraries, or the antibodies can be used for lmmunoscreening following hybrid selection and translation (see, for example. Ausubel ( 1995) at pages 6-12 to 6-16. Margohs et al, "Screening λ expression libraries with antibody and protein probes," in DNA Cloning 2. Expression Svstems, 2nd Edition. Glover et al. (eds.). pages 1-14 (Oxford University Press 1995)) As an alternative, a Ztryp3 gene can be obtained by synthesizing nucleic acid molecules using mutually priming long oligonucleotides and the nucleotide sequences described herein (see, for example. Ausubel ( 1995) at pages 8-8 to 8-9) Established techniques using the polymerase chain reaction provide the ability to synthesize DNA molecules at least two kilobases in length (Adang et al.. Plant Molec Biol. 27.1 131 ( 1993). Bambot et al.. PCR Methods and Applications 2.26b ( 1993 ). Dillon et al.. "Use of the Polymerase Chain Reaction for the Rapid Construction of Synthetic Genes.^" in Methods in Molecular Biologx Vol 15 PCR Protocols Cm rent Methods and Applications. White (ed.), pages 263-268, (Humana Press, Inc. 1993 ). and Holowachuk et al, PCR Methods Appl. 4:299 (1995))

The nucleic acid molecules of the present invention can also be synthesized with "gene machines" using protocols such as the phosphoramidite method. If chemically-synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 base pairs) is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. For the production of longei genes (>300 base pairs), however, special strategies may be required, 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. For reviews on polynucleotide synthesis, see, for example, Glick and Pasternak, Molecular Bιotechnolog\, Principles and Applications of Recombinant DNA (ASM Press 1994). Itakura et al, Anna. Rev. Biochem. 53:323 (1984). and Chmie et al, Proc. Nat 'I Acad Sci. USA 57:633 (1990) The sequence of a Ztryp3 cDNA or Ztryp3 genomic fragment can be determined using standard methods. Ztryp3 polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a Ztryp3 gene. Promoter elements from a Ztryp3 gene can be used to direct the expression of heterologous genes in. for example, testicular tissue of transgemc animals or patients undergoing gene therapy The identification of genomic fragments containing a Ztnp3 promoter or regulatory element can be achieved using well-established techniques, such as deletion analysis (see, generally. Ausubel (1995)) Cloning of 5' flanking sequences also facilitates production of Ztrypi proteins by "gene actuation,^" as disclosed in U S Patent No 5.641.670 Briefly , expression of an endogenous Ztr\p3 gene in a cell is altered by introducing into the Ztryp3 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 Ztnp3 5 non-coding sequence that permits homologous recombination of the construct with the endogenous Ztr\p3 locus, whereby the sequences within the construct become operably linked with the endogenous

coding sequence In this way . an endogenous Ztryp3 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression

4. Production of Ztryp3 Variants

The present invention prov ides a v riety of nucleic acid molecules including DNA and RNA molecules that encode the Ztryp3 polypeptides disclosed herein 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 nucleotide sequence that encompasses all nucleic acid molecules that encode the Ztryp3 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, the present invention contemplates Ztryp3 polypeptide-encoding nucleic acid molecules comprising nucleotides 42 to 746 of SEQ ID NO 1 , and then RNA equivalents

Table 1 sets forth the one-lettei codes used within SEQ ID NO 3 to denote degenerate nucleotide positions "Resolutions^' are the nucleotides denoted by a code lettei "Complement" indicates the code for the complementary nucleotιde(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

Table J

The degenerate codons used in SEQ ID NO:3. encompassing all possible codons for a given amino acid, are set forth in Table 2.

Table 2

One of ordinary skill in the art ill appreciate that some ambiguity is introduced in determining a degenerate codon representative of all possible codons encoding an 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 phenylalanme and leucine Thus some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences but one of ordinarv skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO 2 Variant sequences can be readily tested for

} ( functionality as described herein

Different species can exhibit ' preferential codon usage " In general see. Grantham et al Nuc Acids Res 5 1893 ( 1980) Haas et al Citi i Biol 6 315 ( 1996) W ain-Hobson et al Gene 13 355 (1981 ) Grosiean and Fiers Gene 18 199 ( 1982) Holm, Nuc Acids Res 14 3075 (1986). Ikemura J Mol Biol 158 573 ( 1982). Sharp

15 and Matassi, Cutt Optn Genet

4 851 (1994), Kane. Cuπ Opm Biotechnol 6 494 (1995), and Makπdes, Microbwl Re\ 60 512 (1996) As used herein, the term ' preferential codon usage" or ' preferential codons^" is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid

20 (see Table 2) 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 iruses or bacteria, different threonine codons may be preferential Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a v ariety of methods 5 known in the art Introduction of preferential 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 3 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and 0 disclosed herein Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein

The present invention further provides variant polypeptides and nucleic acid molecules that represent counterparts fiom other species (orthologs) These species include but are not limited to mammalian, avian. amphibian, reptile, fish, insect 5 and other vertebrate and invertebrate species Of particular interest are Ztryp3 polypeptides from other mammalian species, including porcine, muπne, ovine, bovine, canine, feline, equine, and other primate polypeptides Orthologs of human Ztryp3 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 Ztryp3 as disclosed herein. Suitable sources of mRNA can be identified by probing northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell line.

A Ztryp3-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 polymerase chain reaction with primers designed from the representative human Ztryp3 sequences 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 Ztryp3 polypeptide. Similar techniques can also be applied to the isolation of genomic clones. Those skilled in the art will recognize that the sequence disclosed in

SEQ ID NO: l represents a single allele of human Ztryp3, and that allelic variation and alternative splicing are 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 nucleotide sequence shown in SEQ ID NO: l , 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 NO:2. cDNA molecules generated from alternatively spliced mRNAs. which retain the properties of the Ztryp3 polypeptide 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 standard procedures known in the art.

Within certain embodiments of the invention, the isolated nucleic acid molecules can hybridize under stringent conditions to nucleic acid molecules comprising nucleotide sequences disclosed herein. For example, such nucleic acid molecules can hybridize under stringent conditions to nucleic acid molecules comprising the nucleotide sequence of nucleotides 42 to 746 of SEQ ID NO: l. to nucleic acid molecules consisting of the nucleotide sequence of SEQ ID NO: l . to nucleic acid molecules comprising the nucleotide sequence of nucleotides 99 to 746 of SEQ ID NO: l , or to nucleic acid molecules consisting of a nucleotide sequence complementary to nucleotides 42 to 746 of SEQ ID NO:l , nucleotides 99 to 746 of SEQ ID NO: l, or to SEQ ID NO: l . In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe

A pair of nucleic acid molecules, such as DNA-DNA. RNA-RN A and ^ DNA-RNA can hybridize if the nucleotide sequences hav e some degree of complementarity Hybrids can tolerate mismatched base pairs in the double helix, but the stability of the hybrid is influenced by the degree of mismatch The T_m of the mismatched hybrid decreases by 1 °C for every 1 -1 5% base pair mismatch Varying the stringency of the hybridization conditions allow s control over the degree of mismatch l that will be present in the hybrid The degiee of stringency incieases as the hybridization tempeiature increases and the ionic strength of the hybridization buffer decreases Stringent hybridization conditions encompass temperatures of about 5-25°C below the T_m of the hybrid and a hybridization buffer having up to 1 M Na^~ Highei degrees of stringency at lower temperatures can be achieved with the addition of

1 formamide which reduces the 1 _nι of the hybrid about 1 °C foi each 1 % formamide in the buffer solution Generally, such stringent conditions include temperatures of 20-70°C and a hybridization buffer containing up to 6x SSC and 0-50% formamide A higher degree of stringency can be achieved at temperatures of from 40-70°C with a hybridization buffer having up to 4x SSC and from 0-50% formamide Highly stringent 0 conditions typically encompass temperatures of 42-70°C with a hybridization buffer having up to lx SSC and 0-50% formamide Different degrees of stringency can be used during hybridization and washing to achieve maximum specific binding to the target sequence Typically, the washes following hybridization are performed at increasing degrees of stringency to remove non-hvbπdized polynucleotide probes from s hybridized complexes

The above conditions are meant to serve as a guide and it is well w ithin the abilities of one skilled in the art to adapt these conditions for use with a particular polypeptide hybrid The T_m for a specific target sequence is the temperature (under defined conditions) at which 50% of the target sequence will hybridize to a perfectly 0 matched probe sequence Those conditions that influence the T_m include, the size and base pair content of the polynucleotide probe the ionic strength of the hvbπdization solution, and the presence of destabilizing agents in the hybridization solution Numerous equations for calculating T_m are kno n in the art. and are specific for DNA. RNA and DNA-RNA hybrids and polynucleotide probe sequences of varying length s (see. for example. Sambrook et al , Moleculai Cloning A Labotaton Manual. Second Edition (Cold Spring Harbor Press 1989). Ausubel et al , (eds ). Current Ptotocols in Moleculai Bwlog\ (John Wiley and Sons. Inc 1987), Berger and Kimmel (eds ) Guide to Moleculai Cloning Techniques (Academic Press. Inc 198⁷ ) and Wetmui Cut Re\ Biochem Mol Biol 26 227 ( 1990)) Sequence anal sis software such as OLIGO 6 0 (LSR, Long Lake, MN ) and Prunei Piemiei 4 0 (Premier Biosoft International, Palo Alto, CA). as well as sites on the Internet are av ailable tools for s analyzing a given sequence and calculating T_nι based on user-defined criteria Such programs can also analyze a given sequence under defined conditions and identify suitable probe sequences Typically, hybridization of longer polv nucleotide sequences, >50 base pans, is performed at temperatures of about 20-25°C below the calculated T_m For smaller probes, <50 base pairs, hybndization is typically carried out at the T_n1 or 5- l o 10°C below This allow s foi the maximum rate of hybridization foi DNA DNA and DNA-RNA hybrids

The length of the polynucleotide sequence influences the rate and stability of hybrid formation Smallei probe sequences o0 base pairs reach equilibrium with complementary sequences lapidly. but mav form less stable hybrids i Incubation times of anywhere fiom minutes to houis can be used to achiev e hybrid formation Longer probe sequences come to equilibrium more slowlv . but form more stable complexes even at lower temperatures Incubations aie allowed to proceed overnight or longei Generally, incubations are carried out for a period equal to thiee times the calculated Cot time Cot time, the time it takes for the polynucleotide 0 sequences to reassociate. can be calculated for a particular sequence by methods known in the art

The base pair composition of polynucleotide sequence ill effect the thermal stability of the hybrid complex, thereby influencing the choice of hybridization temperature and the ionic strength of the hybridization buffei A T pairs aie less stable

25 than G-C pairs in aqueous solutions containing sodium chloride Therefore, the higher the G-C content, the more stable the hybrid Even distribution of G and C residues within the sequence also contribute positively to hybrid stability In addition, the base pair composition can be manipulated to alter the T_πι of a given sequence For example. 5-methyldeoxycytιdιne can be substituted for deoxycytidine and 5-bromodeoxuπdme 0 can be substituted for thymidine to increase the T_m whereas 7-deazz-2^'-deoxyguanosιne can be substituted for guanosme to reduce dependence on T_m

The ionic concentration of the hybridization buffer also affects the stability of the hybrid Hybridization buffers generally contain blocking agents such as Denhardt's solution (Sigma Chemical Co . St Louis. Mo ). denatured salmon sperm 5 DNA, tRNA, milk powders (BLOTTO), heparin or SDS, and a Na^" source, such as SSC (lx SSC 0.15 M sodium chloride, 15 mM sodium citrate) or SSPE (lx SSPE 1 8 M NaCl, 10 mM NaHιP0₄. 1 mM EDTA, pH 7 7) By decreasing the ionic concentration of the buffei , the stability of the hybrid is increased Typically, hybridization buffers contain from between 10 mM - 1 M Na^{^} The addition of destabilizing or denaturing agents such as formamide, tetralkylammomum salts guamdinium cations oi thiocyanate cations to the hybridization solution w ill alter the T_nι of a hybrid Typically , formamide is used at a concentration of up to 50% to allow incubations to be carried out at more convenient and lower temperatures Formamide also acts to reduce non-specific background when using RNA probes

As an illustration, a nucleic acid molecule encoding a variant Ztryp3 polypeptide can be hybridized with a nucleic acid molecule having the nucleotide

10 sequence of nucleotides 99 to 746 of SEQ ID NO 1 (or its complement) at 42°C overnight in a solution compπsing 50% formamide 5xSSC ( lxSSC 0 15 M sodium cnloπde and 15 mM sodium citrate). 50 mM sodium phosphate (pH 7 6), 5x Denhardt s solution (

Denhardt's solution 2%

i Ficoll 400 2% (w/v ) polyvinylpyrrolidone. and 2%- (w/v ) bovme serum albumin 10% dextran sulfate, and 20 i μg/ml denatured, sheared salmon sperm DNA One of skill in the art can devise variations of these hybridization conditions For example, the hybridization mixture can be incubated at a higher temperature, such as about 65°C, in a solution that does not contain formamide Moreover, premixed hybridization solutions are available (e g EXPRESSHYB Hybridization Solution from CLONTECH Laboratories. Inc ), and 0 hybndization can be performed according to the manufacturer's instructions

Following hybridization, the nucleic acid molecules can be washed to remove non-hybridized nucleic acid molecules undei stringent conditions, or under highly stringent conditions Typical stringent washing conditions include washing in a solution of 0 5x - 2x SSC with 0 1 % sodium dodecyl sulfate (SDS) at 55 - 65°C That 5 is, nucleic acid molecules encoding a variant Ztryp3 polypeptide remain hybridized following stringent washing conditions with a nucleic acid molecule having the nucleotide sequence of nucleotides 99 to 746 of SEQ ID NO 1 (or its complement), in which the wash stringency is equivalent to 0.5x 2x SSC with 0 1 % SDS at 55 - 65°C including 0 5x SSC with 0 1 % SDS at 55°C. or 2xSSC w ith 0 1 % SDS at 65°C One of 0 skill in the art can readily devise equivalent conditions for example, by substituting the SSPE for SSC in the wash solution

Typical highly stringent washing conditions include washing in a solution of 0 lx - 0 2x SSC with 0 1 % sodium dodecyl sulfate (SDS) at 50 - 65°C In other words, nucleic acid molecules encoding a variant Ztryp3 polypeptide remain 5 hybridized following highly stringent washing conditions with a nucleic acid molecule having the nucleotide sequence of nucleotides 99 to 746 of SEQ ED NO 1 (or its complement), in which the wash stringency is equivalent to 0 lx - 0 2x SSC with 0 1 % 7

SDS at 50 - 65°C. including 0 l x SSC with 0 1 % SDS at 50°C, or 0 2xSSC with 0 1 % SDS at 65°C

The present invention also provides isolated Ztryp3 polypeptides that hav e a substantially similar sequence identity to the poly peptide of SEQ ID NO 2 or orthologs The term "substantially similar sequence identity ' is used herein to denote polypeptides having 707c, 807c, 907c 957c 967c 97%. 98% or 997c sequence ιdentιt\ to the sequence shown in SEQ ID NO 2

The present invention also contemplates Ztryp3 v ariant nucleic acid molecules that can be identified using two criteria a determination of the similarity betw een the encoded polypeptide with the amino acid sequence of SEQ ID NO 2 and a hvbπdization assay, as descπbed abov e Such Ztryp3 v ariants include nucleic acid molecules ( 1 ) that remain hybridized follow ing stringent washing conditions w ith a nucleic acid molecule having the nucleotide sequence of nucleotides 99 to 746 of SEQ ID NO 1 (or its complement), in which the wash stringency is equiv alent to 0 5\ - 2x SSC with 0 1 c SDS at 55 - 65°C. and (2) that encode a polypeptide having 709c 807c 90% 957c 96%, 97%, 98%> or 99% sequence identity to the amino acid sequence of SEQ ID NO 2

Alternatively , Ztryp3 variants can be characterized as nucleic acid molecules (1 ) that remain hybridized following highly stringent washing conditions with a nucleic acid molecule having the nucleotide sequence of nucleotides 99 to 746 of SEQ ID NO 1 (or its complement), in which the wash stringency is equivalent to 0

- 0 2x SSC with 0 17c SDS at 50 - 65°C. and (2) that encode a polypeptide having 707c, 807c, 907c. 957c, 967c 977c 987c oi 997c sequence identitv to the amino acid sequence of SEQ ID NO 2 The present invention also includes Ztryp3 variants that possess serine protease enzyme activity Moreover, particular Ztryp3 variants aie characterized using hybridization analysis with a reference nucleic acid molecule that is a fragment of a nucleic acid molecule consisting of the nucleotide sequence of nucleotides 99 to 746 of SEQ ID NO 1. or its complement Percent sequence identity is determined by conventional methods See for example, Altschul et al Bull Math Bio 48 603 ( 1986) and Hemkoff and Hemkoff, Proc Nat 'I Acad Sci USA 89 10915 ( 1992) Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penaltv of 10 a gap extension penalty of 1. and the "BLOSUM62' scoring matrix of Hemkoff and Hemkoff (ibid ) as shown in Table 3 (amino acids are indicated by the standard one- letter codes) The percent identity is then calculated as ([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] )(100).

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< κ 2 Q o ω 5 a: M _q b-: W fπ S > Those skilled in the ait appreciate that theie are many established algorithms available to align tw o amino acid sequences The FASTA^' similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative Ztryp3 v ariant The FASTA algorithm is described by Pearson and Lipman Pioc Nat 'I Acad Sci USA 85 2444 (1988), and by Peaison. Meth Enzxmol 183 63 (1990) Briefly. FASTA first characterizes sequence similaπtv by identifying regions shared by the query sequence (e t> SEQ ID NO 2) and a test sequence that have either the highest density of identities (if the ktup variable is 1 ) oi pans of identities (if ktup=2). without considering conservative amino acid substitutions, insertions oi deletions The ten iegions w ith the highest density oi identities are then rescored bv comparing the similarity of all paned amino acids using an amino acid substitution matπ\. and the ends of the regions aie trimmed to include only those iesidues that contribute to the highest score If there are several regions with scores greater than the "cutoff value (calculated by a piedetermined 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, S1AM J Appl Math 26 787 (1974)), which allow s for amino acid insertions and deletions Illustrativ e parameteis for FASTA analysis are ktup=l , gap opening penalty=10, gap extension penalty=l , and substitution matπx=BLOSUM62 These parameters can be introduced into a FASTA progiam by modifying the scoring matrix file ("SMATRLX") as explained in Appendix 2 of Pearson, Meth

183 63 ( 1990)

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. preferablv from three to six, most preferably three, with other paiameters set as described abov e The present invention includes nucleic acid molecules that encode a polypeptide having a conservative amino acid change, compared w ith the amino acid sequence of SEQ ID NO 2 That is. variants can be obtained that contain one or more amino acid substitutions of SEQ ID NO 2, in which an alkvl amino acid is substituted for an alkyl amino acid in a Ztryp3 ammo acid sequence an aromatic amino acid is substituted for an aromatic ammo acid in a Ztryp3 amino acid sequence, a sulfur- containing amino acid is substituted for a sulfur-containing amino acid in a Ztryp3 amino acid sequence, a hydroxv -containing ammo acid is substituted for a hydroxy - containing ammo acid in a Ztryp3 amino acid sequence, an acidic amino acid is substituted for an acidic amino acid in a Ztryp3 amino acid sequence, a basic amino acid is substituted for a basic amino acid in a Ztryp3 amino acid sequence, oi a dibasic monocarboxylic amino acid is substituted for a dibasic monocarboxyhc amino acid in a Ztryp3 amino acid sequence

Among the common amino acids, for example, a "conservativ e amino acid substitution^" is illustrated by a substitution among amino acids within each of the following groups (1 ) glycme. alanme. va ne. leucine, and isoleucine. (2) phenylalanine, tyrosme, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate. (5) glutamine and asparag e. and (6) lysine. arginine and histidine

The BLOSUM62 table is an amino acid substitution matrix deriv ed fiom about 2.000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Hemkott and Hemkoff. Proc. Nat 7 Acad. Sci. USA 89: 10915 ( 1992)) Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed above), the language "conservative amino acid substitution" preferably refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1 , 2, or 3 According to this system, preferred consen ative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g , 1. 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3 ) Particular variants of Ztryp3 are characterized by having greater than

96%, at least 97%, at least 98%, or at least 997c sequence identity to the corresponding amino acid sequence (e.g., SEQ ID NO:2), wherein the variation in amino acid sequence is due to one or more conservative amino acid substitutions.

Conservative amino acid changes in a Ztrvp3 gene can be introduced by substituting nucleotides for the nucleotides recited in SEQ ID NO: l Such "conservative amino acid" variants can be obtained, foi example, by ohgonucleotide- directed mutagenesis, linker-scanning mutagenesis. mutagenesis using the polymerase chain reaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22; and McPherson (ed.). Directed Mutagenesis: A Practical Approach (IRL Press 1991 )). The proteins of the present invention can also comprise non-naturally occurring am o acid residues. Non-naturally occurring amino acids include, without limitation, trø/.i-3-methylprolme, 2.4-methanoprolιne. c/5-4-hydroxyprohne, trans-4- hydroxyproline, N-methylglycine. α//othreonine, methylthreonine. hydroxyethylcysteine. hydroxyethylhomocysteme. nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline. 3.3-dimethylproIine, tert-leucine, norvaline. 2-azaphenylalanine. 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine. Several methods are know n in the art for incorporating non-naturally occuπ'ing 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 typically carried out in a cell-free system comprising an E. coll 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 ). Εllman et l.. Methods Enzymol. 202:301 ⁽ 1991 ). Chung et al, Science 259:806 ( 1993), and Chung et al, Proc. Nat ^'l Acad. Sci. USA 9(9: 10145 ( 1993).

In a second method, translation is carried out in Xenopits oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al, J. Biol Chem. 271: 19991 ( 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 ( 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 ( 1993)).

A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for Ztryp3 amino acid residues.

Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells. Science 244: 1081 (1989). Bass et al. Proc. Nat'l Acad. Sci. USA 88:4498 (1991). Coombs and Corey. "Site- Directed Mutagenesis and Protein Engineering," in Proteins: Analysis and Design, Angeletti (ed.), pages 259-31 1 (Academic Press. Inc. 1998)). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the _ -), - s,

resultant mutant molecules aie tested foi biological activitv as disclosed below to identify ammo acid lesidues that are critical to the activity of the molecule See also. Hilton et al J Biol Chem 271 4699 ( 1996) The identities of essential ammo acids can also be inferred from analysis of homologies with other serine proteases The location of Ztryp3 activ ity domains can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids See for example, de Vos <?t al Science 255 306 (1992), Smith et al . J Mol Biol 224 899 ( 1992 ). and Wlodaver et al FEBS Lett 309 59 ( 1992) Moreovei, Ztryp3 labeled ith biotin or FfTC can be used for expression cloning of Ztryp3 substrates and inhibitors

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening such as those disclosed bv Reidhaar-Olson and Sauer (Science 241 53 ( 1988)) or Bowie and Sauer (Pioc hat I Acad Sci USA 56.2152 (1989)) Briefly , these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagemzed 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 ( 1991 ). Ladner et al U S Patent No 5.223.409, Huse, international publication No WO 92/06204 and region-directed mutagenesis (Derbyshne et al Gene 46 145 ( 1986), and Ner et al DNA 7. 127, (1988))

Variants of the disclosed Ztryp i nucleotide and polypeptide sequences can also be generated through DNA shuffling as disclosed by Stemmer. Natiue 370 389 (1994), Stemmer, Proc Nat l Acad Sci USA 91 10747 (1994), and international publication No WO 97/20078 Briefly, variant DNAs are generated by in \ ιtro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR. resulting in randomly introduced point mutations This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs 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

Mutagenesis methods as disclosed herein can be combined with high- throughput, automated screening methods to detect activity of cloned, mutagemzed polypeptides in host cells Mutagemzed DNA molecules that encode biologically active polypeptides. or polypeptides that bind with antι-Ztryp3 antibodies, can be recov ered from the host cells and rapidly sequenced using modern equipment These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure

The present invention also includes "functional fragments^" of Ztryp3 s polypeptides and nucleic acid molecules encoding such functional fragments Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a Ztryp3 polypeptide As an illustration. DNA molecules having the nucleotide sequence of SEQ ID NO 1 can be digested with Ba!31 nuclease to obtain a series of nested deletions One alternative to 0 exonuclease digestion is to use ohgonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production ot a desired fragment Alternatively, particular fragments of a Ztr\p3 gene can be synthesized using the polymeiase chain reaction

As an illustration of this general approach, studies on the truncation at 5 either or both termini of interferons have been summarized by Hoπsberger and Di Marco, Pharmac. Ther 66:507 (1995) Moreover, standard techniques for functional analysis of proteins are described by, for example, Treuter et al , Molec Gen Genet 240.1 13 (1993), Content et al , "Expression and preliminary deletion analysis of the 42 kDa 2-5 A synthetase induced by human interferon," in Biological Interferon S\ stems, 0 Proceedings of 1SIR-TNO Meeting on Interferon Sxstems. Cantell (ed ), pages 65-72 (Nijhoff 1987), Herschman, "The EGF Receptoi." in Control oj Animal Cell Proliferation, Vol. 1. Boynton et al, (eds ) pages 169-199 (Academic Press 1985), Coumailleau et al, J Biol Chem. 270:29270 ( 1995). Fukunaga et al, J Biol Chem 270:25291 ( 1995); Yamaguchi et al, Biochem Pharmacol 50.1295 (1995 ), and Meisel 5 et al, Plant Molec. Biol 30.1 (1996)

The present invention also contemplates functional fragments of a Ztryp3 gene that has amino acid changes, compared with the amino acid sequence of SEQ ID NO.2 A variant Ztryp3 gene can be identified on the basis of structure by determining the level of identity with nucleotide and amino acid sequences of SEQ ID 0 NOs: l and 2, as discussed above An alternative approach to identifying a variant gene on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant Ztnp3 gene can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO.1, as discussed above

The present invention also provides polypeptide fragments or peptides 5 comprising an epitope-beaπng portion of a Ztryp3 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-beaπng peptides can be identified using standard methods (see. for example. Geysen et al, Proc Nat 'l Acad Sci I SA 81 3998 (1983))

In contrast, polypeptide fragments or peptides may comprise an ' antigemc epitope." which is a region of a protein molecule to w hich 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. Sutc ffe et al , Science 219:660 (1983)) Accordingly, antigemc epitope- bearing peptides and polypeptides of the present invention are useful to raise antibodies that bind with the polypeptides described herein

Antige c epitope-beaπng peptides and polypeptides can contain at least four to ten ammo acids, at least ten to fifteen amino acids, or about 15 to about 30 amino acids of SEQ ID NO.2 Such epitope-beaπng peptides and polypeptides can be produced by fragmenting a Ztryp3 polypeptide. or by chemical peptide synthesis, as descπbed 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. Bwtechnol 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 Biologx, Vol 10. Manson (ed.), pages 105-1 16 (The Humana Press. Inc 1992), Price, "Production and Characterization of Synthetic Peptide-Deπved 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 Immunologx . pages 9.3.1 - 9.3.5 and pages 9.4.1 - 9.4.1 1 (John Wiley & Sons 1997)

For any Ztryp3 polypeptide, including variants and fusion proteins, one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above. Moreover, those of skill in the art can use standard software to devise Ztryp3 variants based upon the nucleotide and ammo acid sequences described herein Accordingly, the present invention includes a computer-readable medium encoded with a data structure that provides at least one of SEQ ID NO. l, SEQ ID NO.2. and SEQ ID NO 3 Suitable forms of computer-readable media include magnetic media and optically- readable media. Examples of magnetic media include a hard or fixed drive, a random access memory (RAM) chip, a floppy disk, digital linear tape (DLT), a disk cache, and a ZIP disk Optically readable media are exemplified by compact discs (e g.. CD-read onlv memory (ROM) CD-rewritable (RW) and CD-recordable) and digital versatile/video discs (DVD) (e g DVD-ROM DVD-RAM, and DVD+RW)

5. Production ofZtryp3 Fusion Proteins Fusion proteins of Ztryp3 can be used to express Ztryp3 in a recombinant host, and to isolate expressed Ztιyp3 One type of fusion protein comprises a peptide that guides a Ztryp3 polypeptide from a recombinant host cell To direct a Ztryp3 polypeptide into the secretory pathway of a eukaryotic host cell a secretoiy signal sequence (also known as a signal peptide. a leadei sequence, prepio sequence or pre sequence) is provided in the Ztιyp expression vector While the secretoiy signal sequence may be derived from Ztryp3. a suitable signal sequence

also be derived from another secreted piotein or svnthesized de no\ υ The secretorv signal sequence is operably linked to a Ztryp3-encodιng sequence such that the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathwav of the host cell Secretory signal sequences are commonly positioned 5 to the nucleotide sequence encoding the polypeptide of interest, although certain secretoiy signal sequences may be positioned elsewhere in the nucleotide 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) While the secretory signal sequence of Ztryp3 oi anothei protein produced by mammalian cells (e g , tissue type plasminogen activator signal sequence, as described, for example, in U S Patent No 5 641 655) is useful for expression of Ztryp3 in recombinant mammalian hosts, a yeast signal sequence is preferred for expression in yeast cells Examples of suitable yeast signal sequences are those derived from yeast mating phermone α- factor (encoded by the MFal gene), invertase (encoded by the SUC2 gene), or acid phosphatase (encoded by the PH05 gene) See for example, Romanos et al , "Expression of Cloned Genes in Yeast,^" in DNA Cloning 2 A Practical Approach, 2^nd Edition, Glover and Hames (eds ), pages 123-167 (Oxford University Press 1995) In bacterial cells, it is often desirable to express a heterologous protein as a fusion protein to decrease toxicity, increase stability, and to enhance recoveiy of the expressed protein For example, Ztryp3 can be expressed as a fusion protein comprising a glutathione S-transferase polypeptide Glutathione S-transfeiease fusion proteins are typically soluble, and easily purifiable from E coli lysates on immobilized glutathione columns In similar approaches, a Ztryp3 fusion protein comprising a maltose binding protein polypeptide can be isolated with an amylose resm column M

while a fusion protein comprising the C-terminal end of a truncated Protein A gene can be purified using IgG-Sepharose Established techniques for expressing a heterologous polypeptide as a fusion protein in a bacterial cell are described, for example, by Williams et al "Expression of Foieign Pioteins in E coli Using Plasmid Vectois and Purification of Specific Polvclonal Antibodies ' in DNA Cloning 2 A Practical Approach, 2^nd Edition, Glover and Hames (Eds ), pages 15-58 (Oxford University Press 1995) In addition, commercially available expression systems are available For example, the PINPOINT Xa protein purification system (Promega Corporation, Madison WI) provides a method for isolating a fusion protein comprising a polypeptide that becomes biotinylated during expression with a resin that comprises avidin

Peptide tags that aie useful for isolating heterologous polypeptides expressed by either prokaryotic or eukaivotic cells include polvHistidine tags (which have an affinity for mckel-chelatmg resin ) c-nnc tags calmoduhn binding protein (isolated with calmoduhn affinity chromatography ). substance P the RYIRS tag (which binds with anti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which binds with anti-FLAG antibodies) See, for example. Luo et al , Arch Biochem Bioplns 329.215 (1996), Morganti et al , Bwtechnol Appl Biochem 23 67 (1996), and Zheng et al , Gene 186.55 (1997) Nucleic acid molecules encoding such peptide tags are available, for example, from Sigma-Aldπch Corporation (St Louis, MO) Another form of fusion protein comprises a Ztryp3 polypeptide and an immunoglobuhn heavy chain constant region typically an F_c fragment which contains two constant region domains and a hinge region but lacks the v ariable region As an illustration, Chang et al . U S Patent No 5,723,125, describe a fusion protein comprising a human interferon and a human immunoglobuhn Fc fragment, in which the C-terminal of the interferon is linked to the N-terminal of the Fc fragment by a peptide linker moiety An example of a peptide linker is a peptide comprising primarily a T cell inert sequence, which is immunologically inert An exemplary peptide linker has the amino acid sequence GGSGG SGGGG SGGGG S (SEQ ID NO 4) In such a fusion protein, an illustrative Fc moiety is a human γ4 chain, which is stable in solution and has little or no complement activating activity Accordingly the present invention contemplates a Ztryp3 fusion protein that comprises a Ztryp3 moiety and a human Fc fragment, wherein the C-terminus of the Ztryp3 moiety is attached to the N-terminus of the Fc fragment via a peptide linker, such as a peptide consisting of the amino acid sequence of SEQ ID NO 4 The Ztιyp3 moiety can be a Ztryp3 molecule or a fragment thereof

In another variation, a Ztryp3 fusion protein comprises an IgG sequence, a Ztryp3 moiety covalently joined to the aminoterminal end of the IgG sequence, and a signal peptide that is covalently |oιned to the aminoteiminal of the Ztryp3 moiety wherein the IgG sequence consists of the following elements in the following order, a hinge region, a CHo domain, and a CH^ domain Accoidmgly. the IgG sequence lacks a CH] domain The Ztryp3 moiety displays a Ztryp3 activitv . as described herein, such as s the ability to bind with a Ztryp3 antibody This general approach to producing fusion piOteins that comprise both antibody and nonantibody portions has been described by LaRochelle et al. EP 742830 (WO 95/21258)

Fusion proteins comprising a Ztryp3 moiety and an Fc moiety can be used, for example, as an //; vitro assay tool For example, the presence of a Ztryp3

10 substrate or inhibitoi in a biological sample can be detected using a Ztryp3-antιbodv fusion protein, in which the Ztryp3 moiety is used to target the substrate or inhibitoi and a macromolecule. such as Protein A or anti-Fc antibody is used to detect the bound fusion piotein-receptor complex Furthermore such fusion proteins can be used to identify molecules that interfere w ith the binding of Ztryp3 and a substrate i Moreover, using methods described in the art. hybrid Ztryp3 proteins can be constructed using regions or domains of the inventive Ztryp3 in combination with those of other serine proteases (e.g trypsin, tryptase. kalhkrein, chymotrypsin, subtihsin, prostate specific antigen, chymotryptic enzyme of skin, and protease M. etc ). or heterologous proteins (see, for example, Picard, Cur Opin. Bιolog\ 5:51 1 (1994)) 0 These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide. and can be applied to polypeptides of unknown structure For example Hoπsberger and DiMarco, Pharmac Thei 66 507 (1995). describe the 5 construction of fusion protein hybrids comprising different interferon-α subtypes, as well as hybrids comprising interferon-α domains from different species

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 the components Alternatively, a polynucleotide encoding both components of the 0 fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein General methods for enzymatic and chemical cleavage of fusion proteins are described, foi example, by Ausubel (1995) at pages 16-19 to 16-25 6. Ztryp3 Analogs and Ztryp3 Inhibitors

One general class of Ztryp3 analogs are v aπants having an amino acid sequence that is a mutation of the amino acid sequence disclosed herein Another general class of Ztryp3 analogs is provided bv anti-idiotype antibodies, and fragments thereof, as described below Moreov er, recombinant antibodies comprising anti- ldiotype variable domains can be used as analogs (see. for example, Monfardim et αl Proc Assoc Am Plnsiciαns 108 420 ( 1996)) Since the variable domains of anti- ldiotype Ztryp3 antibodies mimic Ztryp3, these domains can provide Ztryp3 enzymatic activity Methods of producing anti-idiotypic catalytic antibodies are known to those of skill in the art (see. for example. Joron <?t αl Ann N Y Acαd Sci 672 216 ( 1992 ), Fπboulet et αl , Appl Biochem Biotechnol 47 229 ( 1994), and Avalle et αl Ann V }

Anothei approach to identifying Ztryp3 analogs is provided bv the use of combinatorial libraries Methods for constiucting and screening phage displav and other combinatorial libraries are provided, for example, by Kay et αl , Phαge Dispkn of Peptides and Proteins (Academic Press 1996) Verdine. U S Patent No 5,783 384. Kay. et al , U.S Patent No 5.747,334, and Kauffman et al , XX S Patent No 5,723.323

One illustrative in vitro use of Ztryp3 and its analogs is the production of labeled peptides from a labeled protein substrate A serine protease, like Ztryp3 can also be used in detergents and cleaning solutions For example, serine proteases are used in solutions to clean and to disinfect contact lenses (see. for example, Aaslyng et A/ L S Patent No 5,985,629) Another use for a serine protease like Ztryp3 is in the formulation of vaccines (see, for example, Dalton et a/ . U S Patent No 5.885.814) A protease like Ztryp3 can also be used to as a reagent to dissociate adherent cells from tissue culture plates Those of skill in the art can devise other uses for molecules having Ztryp3 activity

The activity of Ztryp3 molecules of the present invention can be measured using a variety of assays that measure serine protease activity For example. Ztryp3 enzyme activity can be assessed by a standard in \ ιtιo serine protease assay ( see. for example, Stief and Heimburger. U S Patent No 5,057,414 (1991 )) Those of skill in the art are aware of a variety of substrates suitable for in vitro assays, such as Suc- Ala-Ala-Pro-Phe-pNA, Bz-Val-Gly-Arg-pNA-AcOH. fluorescein mono-p- guanidinobenzoate hydrochloride. benzyloxycarbonyl-L-Arginyl-S-benzylester. Nalpha- Benzoyl-L-argimne ethv l ester hydrochloride. and the like In addition, protease assay kits available from commercial sources, such as Cal biochem® (San Diego, CA) For general references, see Barrett (Ed ). Methods in

Proteohtic Enzxmes Serine and Cxsteine Peptidases (Academic Press Inc 1994), and Barrett et al , (Eds ), Handbook of Proteolytic Enzymes (Academic Press Inc 1998)

Solution in vitio assays can be used to identify a Ztryp3 substrate or inhibitor Solid phase systems can also be used to identify a substrate or inhibitor of a Ztryp3 polypeptide For example, a Ztryp3 polypeptide or Ztryp3 fusion protein can be immobilized onto the surface of a receptor chip of a commercially available biosensor instrument (BIACORE. Biacore AB. Uppsala. Sweden) The use of this instrument is disclosed, for example, by Karlsson. Immunol Methods 145.229 (1991 ). and Cunningham and Wells, J Mol Biol 234 554 ( 1993) In brief, a Ztryp3 polypeptide or fusion protein is cov alently attached using amine or sulfhydryl chemistry , to dextran flbei s that are attached to gold film within a flow cell A test sample is then passed through the cell It a Ztryp3 substrate or inhibitor is present in the sample, it will bind to the immobilized polypeptide or fusion protein, causing a change in the refractiv e index of the medium which is detected as a change in surface plasmon resonance of the gold film This system allows the determination on- and off-rates, from which binding affinity can be calculated, and assessment of the stoichiometry of binding, as well as the kinetic effects of Ztryp3 mutation This system can also be used to examine antibody-antigen interactions, and the interactions of other complement/anti-complement pairs Accordingly, polypeptides of the present invention are useful as targets for identifying modulators of serine protease activity More particularly . Ztryp3 polypeptides are useful for screening or identifying new serine protease inhibitors

7. Production of Ztryp3 Polypeptides in Cultured Cells The polypeptides of the present invention, including full-length polypeptides, functional fragments, and fusion proteins, can be produced in recombinant host cells following conventional techniques To express a Ztrvp3 gene, a nucleic acid molecule encoding the polypeptide must be operably linked to regulatory sequences that control transcπptional expression in an expression vector and then, introduced into a host cell In addition to transcnptional regulatory sequences, such as promoters and enhancers, expression vectors can include translational regulatory sequences and a marker gene which is suitable for selection of cells that carry the expression vector

Expression vectors that are suitable for production of a foreign protein in eukaryotic cells typically contain (1 ) prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in a bacterial host, (2 ) eukaryotic DNA elements that control initiation of transcription, such as a promoter, and (3) DNA elements that control the processing of transcripts, such as a transcription termmation/polyadenylation sequence. As discussed above, expression vectors can also include nucleotide sequences encoding a secretory sequence that directs the heterologous polypeptide into the secretory pathway of a host cell For example, a Ztryp3 expression vector may comprise a Ztr\p3 gene and a secretory sequence derived from a Ztr\p3 gene or another secreted gene.

Ztryp3 proteins of the present invention may be expressed in mammalian cells Examples of suitable mammalian host cells include African green monkey kidney cells (Vero, ATCC CRL 1587). human embryonic kidney cells (293-HEK. ATCC CRL 1573). baby hamster kidney cells (BHK-21. BHK-570. ATCC CRL 8544. ATCC CRL 10314). canine kidney cells (MDCK. ATCC CCL 34 ). Chinese hamster ovary cells (CHO-K1. ATCC CCL61 ; CHO DG44 (Chasm et al Som Cell Molec Genet. 12:555, 1986)). rat pituitary cells (GH1. ATCC CCL82), HeLa S3 cells (ATCC CCL2.2). rat hepatoma cells (H-4-LT-E; ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650) and muπne embryonic cells (NIH-3T3; ATCC CRL 1658).

For a mammalian host, the transcπptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression Suitable transcπptional and translational regulatory sequences also can be obtained from mammalian genes, such as actin, collagen, myosin, and metallothionein genes

Transcπptional regulatory sequences include a promoter region sufficient to direct the initiation of RNA synthesis Suitable eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer et al, J. Molec Appl Genet. 1:273 (1982)), the TK promoter of Herpes virus (McKnight, Cell 31.355 (1982)). the SV40 early promoter (Benoist et al . Nature 290.304 (1981)). the Rons sarcoma virus promoter (Gorman et al, Proc. Nat'l Acad. Sci. USA 79.6777 (1982)). the cytomegalovirus promoter (Foecking et al, Gene 45: 101 (1980)), and the mouse mammary tumor virus promoter (see, generally. Etcheverry. "Expression of Engineered Proteins in Mammalian Cell Culture," in Protein Engineering- Principles and Practice. Cleland et al (eds.), pages 163-181 (John Wiley & Sons, Inc. 1996))

Alternatively, a prokaryotic promoter, such as the bacteπophage T3 RNA polymerase promoter, can be used to control Ztryp3 gene expression in mammalian cells if the prokaryotic promoter is regulated by a eukaryotic promoter ,_«,,, PCT/US01/06432 01/66771

42

(Zhou et al , Mol Cell Biol 10 4529 ( 1990). and Kaufman et al Nucl Acids Res 79 4485 ( 1991 ))

An expression vector can be introduced into host cells using a variety of standaid techniques including calcium phosphate transfection posome-mediated transfection. microprojectile-mediated delivery electroporation, and the like The transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome Techniques for introducing vectors into eukaryotic cells and techniques for selecting such stable transformants using a dominant selectable marker are described for example by Ausubel (1995) and by Murray (ed ), Gen Tran sfer and Expr es sion Protocols (Humana Press 1991 )

For example one suitable selectable marker is a gene that pi ov ides lesistance to the antibiotic neomvcin In this case 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 cultuπng transfectants in the presence of a low level of the selectiv e agent and then increasing the amount of selectiv e agent to select for cells that produce high levels of the products of the introduced genes An exemplary amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate Other drug resistance genes (e g hygromyc resistance multi-drug resistance, puromycin acetyltransferase) can also be used Alternatively , markers that introduce an altered phenotype, such as green fluorescent protein, oi cell surface proteins (e g CD4 CD8, Class I MHC and placental alkaline phosphatase) may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology

Ztryp3 polypeptides can also be produced bv cultured cells using a v iral delivery system Exemplary viruses for this purpose include adenovirus, herpesvirus, vaccinia virus and adeno-associated virus (AAV) Adenovirus. a double-stranded DNA virus, is currently the best studied gene transfer v ector for delivery of heterologous nucleic acid (for a review, see Becker et al Meth Cell Biol 43 161 (1994), and Douglas and Cuπel, Science & Medicine 4 44 (1997)) Advantages of the adenov irus system include the accommodation of relatively large DNA inserts, the ability to grow to high-titer, the ability to infect a broad range of mammalian cell types, and flexibility that allows use with a large number of available vectors containing different promoters 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 hgation or by homologous recombination with a co- transfected plasmid An option is to delete the essential El gene from the viral vectoi which results in the inability to iephcate unless the El gene is piovided by the host cell For example, adenovirus vector infected human 293 cells (ATCC Nos CRL 1573 45504 45505) can be grown as adherent cells or in suspension culture at relatu elv high cell density to produce significant amounts of protein (see Gamier et al Cxtotechnol 15 145 (1994))

Zιr\p3 genes may also be expressed in other higher eukaryotic cells such as avian, fungal, insect, yeast, or plant cells The baculovirus system provides an efficient means to introduce cloned

genes into insect cells Suitable expression vectois are based upon the Autographa calif oinica multiple nuclear polvhedrosis virus (AcMNPV). and contain well-know n piomoters such as Diosophila heat shock protein (hsp) 70 promoter Autogr apha califormca nuclear polvhedrosis v irus immediate earlv gene promoter ( e 1) and the delayed early 39K piomoter baculovirus plO promoter and the Diosophila metallothionein promoter A second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow . et al J Virol 674566 (1993)) This system, which utilizes transfer vectors, is sold in the BAC-to-BAC kit (Life Technologies, Rockville, MD) This system utilizes a uansfer vector, PFASTBAC (Life Technologies) containing a Tn7 transposon to move the DNA encoding the Ztryp3 polypeptide into a baculovirus genome maintained in E coli as a large plasmid called a "bacmid " See, Hill-Perkins and Possee, J Gen Vn l 71 971 (1990), Bonning, et al , J Gen Virol 75 1551 ( 1994), and Chazenbalk, and Rapoport J Biol Chem 270 1543 (1995 ) In addition, tianstei vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N -terminus of the expressed Ztryp3 polypeptide. for example, a Glu-Glu epitope tag (Grussenmeyer et al Pioc Nat'l Acad Sci 52 7952 (1985)) Using a technique know n in the art, a transfer vector containing a Ztryp3 gene is transformed into E coli. and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus The bacmid DNA containing the recombinant baculovirus genome is then isolated using common techniques The illustrative PFASTBAC vector can be modified to a considerable degree For example, the polyhedπn promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor . p6 9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins (see, for example Hill-Perkins and Possee, J Gen Virol 71 971 (1990). Bonning, et al , J Gen Vnol 75 1551 (1994), and Chazenbalk and Rapoport, J Biol Chem 270 1543 (1995) In such transfer vector constructs, a short or long version of the basic protein piomoter can be used Moreover, transfei vectors can be constructed which replace the native Ztryp3 secretory signal sequences with secretory signal sequences derived from insect proteins. For example, a secretory signal sequence from Ecdysteroid Glucosyltransfera.se (EGT), honey bee Melittin (Invitrogen Corporation; Carlsbad, CA). or baculovirus gp67 (PharMingen: San Diego. CA) can be used in constructs to replace the native Ztryp3 secretory signal sequence.

The recombinant virus or bacmid is used to transfect host cells. Suitable insect host cells include cell lines derived from IPLB-5/-21. a Spodoptera frugiperda pupal ovarian cell line, such as 5 9 (ATCC CRL 171 1 ), S 21AE, and 5/21 (Invitrogen Corporation; San Diego. CA), as well as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No. 5.300.435 ). Commercially available serum-free media can be used to grow and to maintain the cells. Suitable media are Sf900 II™ (Life Technologies) or ESF 92 l^τr (Expression Systems) for the Sf9 cells: and Ex-cellO405™ (JRH Biosciences. Lenexa. KS i or Express FiveO™ (Life Technologies) for the 79. ni cells. When recombinant virus is used, the cells are typically grown up from an inoculation density of approximately 2-5 x 10 cells to a density of 1-2 x 10⁶ cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.

Established techniques for producing recombinant proteins in baculovirus systems are provided by Bailey et al. "Manipulation of Baculovirus Vectors," in Methods in Molecular Biology, Volume 7: Gene Transfer and Expression Protocols, Murray (ed.), pages 147-168 (The Humana Press. Inc. 1991 ). by Patel et al, "The baculovirus expression system." in DNA Cloning 2: Expression Systems. 2nd Edition. Glover et al. (eds.), pages 205-244 (Oxford University Press 1995). by Ausubel (1995) at pages 16-37 to 16-57. by Richardson (ed. ). Baculovirus Expression Protocols (The Humana Press, Inc. 1995), and by Lucknow, "Insect Cell Expression Technology." in Protein Engineering: Principles and Practice. Cleland et al. (eds.), pages 183-218 (John Wiley & Sons, Inc. 1996).

Fungal cells, including yeast cells, can also be used to express the genes described herein. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Suitable promoters for expression in yeast include promoters from GALl (galactose). PGK (phosphoglycerate kinase). ADH (alcohol dehydrogenase). AOX1 (alcohol oxidase). HIS4 (histidinol dehydrogenase). and the like. Many yeast cloning vectors have been designed and are readily available. These vectors include Yip-based vectors, such as YIp5, YRp vectors, such as YRpl7, YEp vectors such as YEpl3 and YCp vectors, such as YCp 19. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides 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 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). An illustrative vector system for use in Saccharomyces cerevisiae is the PO77 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. Additional suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki. U.S. Patent No. 4.599.31 1, 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, Schizosaccharomyces pornbe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillerrnondii and Candida maltosa are known in the art. See, for example. Gleeson et al, J. Gen. Microbiol 132:3459 (1986), and Cregg, U.S. Patent No. 4.882,279. Aspergillus cells may be utilized according to the methods of McKnight <?t 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.

For example, the use of Pichia methanolica as host for the production of recombinant proteins is disclosed by Raymond, U.S. Patent No. 5.716,808. Raymond. U.S. Patent No. 5,736.383. Raymond et al, Yeast 14: 1 1-23 ( 1998). and in international publication Nos. WO 97/17450, WO 97/17451, WO 98/02536. and WO 98/02565. DNA molecules for use in transforming P. methanolica w ll commonly be prepared as double-stranded, circular plasmids. which can be linearized prior to transformation. For polypeptide production in P. methanolica, it is preferred that the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, the entire expression segment of the plasmid can be flanked at both ends by host DNA sequences. An illustrative selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), and which allows ade2 host cells to grow in the absence of adenine. For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is possible to use host cells in which both methanol utilization genes (AUG1 and AUG2) aie deleted For production of secreted proteins host cells can be used that are deficient in vacuolar protease genes (PEP4 and PRBJ) Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P methanolica cells P methanolica cells can be transformed by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2 5 to 4 5 kV/cm. preferably about 3 75 kV/cm. and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds

Expression vectors can also be introduced into plant protoplasts intact plant tissues, or isolated plant cells Methods for mtioducing expiession vectois into plant tissue include the direct infection oi co cultiv ation of plant tissue with Asrobacteruvn titmefaciens microproiectile mediated delivery DNA miection electroporation. and the like See for example. Horsch et al Science 227 1229 ( 1985). Klein et al , Biotechnology 10 268 (1992). and Miki et al "Piocedures for Introducing Foreign DNA into Plants," in Methods in Plant Molecular Bwlogx and Biotechnology . Ghck et al (eds ), pages 67-88 (CRC Press, 1993)

Alternatively, Ztrxp3 genes can be expressed in prokaryotic host cells Suitable promoters that can be used to express Ztryp3 polypeptides in a prokaryotic host are well-known to those of skill in the art and include promoters capable of recognizing the T4, T3, Sp6 and T7 polymerases, the P and P_L promoters of bacteπophage lambda, the tip, recA, heat shock, lacUVS. tac, Ipp-lacSpr . phoA. and lacZ promoters of E coli promoters of 5 subtilis. the promoters of the bacteriophages of Bacillus, Streptomxces promoteis, the int promoter of bacteπophage lambda the bla promoter of pBR322. and the CAT promoter of the chloramphenicol acetyl transferase gene Prokaryotic promoters have been reviewed by Ghck, J Ind Microbwl 1.277 (1987), Watson et al Molecular Biology of the Gene. 4th Ed (Benjamin Cummins 1987). and by Ausubel et al (1995)

Useful prokaryotic hosts include E coli and Bacillus subtύus Suitable strains of E coli include BL21(DΕ3), BL21(DE3)pLysS. BL21(DE3)pLysE, DH1 , DH4I. DH5, DH5I. DH5IF, DH5IMCR, DH10B, DH10B/p3. DH11S. C600. HB 101. JM101 , JM105, JM109, JM1 10, K38, RR1, Y1088. Y1089, CSH18, ER1451 , and ER1647 (see, for example. Brown (ed ). Moleculai Bwlogx Labfax (Academic Press 1991)) Suitable strains of Bacillus subtilus include BR151. YB886. Mil 19, MI 120, and B170 (see, for example, Hardy, "Bacillus Cloning Methods ^" in DNA Cloning A Practical Approach, Glover (ed ) (ERL Press 1985))

When expressing a Ztryp3 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 aie lysed, and the granules are recovered and denatured using for example, guanidine isothiocyanate or urea The denatured polypeptide can then be refolded and dimeπzed by diluting the denaturant such as bv dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed bv dialv sis 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 (bv for example, somcation or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding Methods for expressing proteins in prokaryotic hosts are well-kno n to those of skill in the art (see for example Williams et al "Expression of foieign proteins in E coli using plasmid vectors and purification of specific polvclonal antibodies ' in DNA Cloning 2 Expiession S stems 2nd Edition Glover et al (eds ) page 15 (Oxford University Press 1995) Ward et al "Genetic Manipulation and Expression of Antibodies,' in Monoclonal Antibodies Pi maples and Applications, page 137 (Wiley-Liss, Inc 1995), and Georgiou "Expression of Proteins in Bacteria," in Protein Engineering Principles and Practice, Cleland et al (eds ), page 101 (John Wiley & Sons, Inc 1996))

Standard methods for introducing expression vectors into bacterial, yeast, insect, and plant cells are provided, for example, by Ausubel (1995)

General methods for expressing and recovering foreign protein produced by a mammalian cell system are provided by for example, Etcheverry ' Expiession of Engineered Proteins in Mammalian Cell Culture ' in Protein Engineering Principles and Pr actice, Cleland et al (eds ), pages 163 (Wiley-Liss Inc 1996) Standard techniques for recovering protein produced by a bacterial system is provided by, for example, Gπsshammer et al , "Purification of over-produced proteins from E coli cells," in DNA Cloning 2 Expression Sxstems. 2nd Edition. Glover et al (eds ), pages 59-92 (Oxford University Press 1995) Established methods for isolating recombinant proteins from a baculovirus system are described by Richardson (ed ) Baculovirus Expression Protocol (The Humana Press, Inc 1995)

As an alternative polypeptides of the present invention can be synthesized by exclusive solid phase synthesis, partial solid phase methods fragment condensation or classical solution synthesis These synthesis methods are well-known to those of skill in the art (see, for example Merπfield, / Am Chem Soc 55 2149 (1963), Stewart et al , "Solid Phase Peptide Synthesis" (2nd Edition) (Pierce Chemical Co 1984), Bayer and Rapp, Chem Pept Prot 3 3 (1986), Atherton et al Solid Phase Peptide Sxnthesis A Practical Approach (IRL Press 1989), Fields and Colowick. "Solid-Phase Peptide Synthesis," Methods in Volume 289 (Academic Press 1997), and Lloyd-Williams et al Chemical Approaches to the Synthesis of Peptides and Proteins (CRC Press, Inc 1997)) Variations in total chemical synthesis strategies such as " native chemical hgation' and " expressed protein hgation^' are also standard (see for example. Dawson et al Science 266 776 (1994) Hackeng et al , Proc Nat I Acad Sci USA 94 7845 (1997), Dawson, Methods Enzxmol 287 34 (1997). Mun et al Proc Nat I Acad Sci USA 95 6705 (1998) and Seveπnov and Mun. J Biol Chem 273 16205 (1998))

8. Isolation of Ztryp3 Polypeptides

T he polypeptides of the present inv ention can be purified to at least about 807c purity, to at least about 909c punty to at least about 95% purity or gieatei than 95% puritv w ith respect to contaminating macromolecules, particulailv other proteins and nucleic acids, and free of infectious and pyrogemc agents The polypeptides of the present invention may also be purified to a phaimaceutically pure state, which is greater than 99 9% pure Certain purified polypeptide preparations are substantially free of other polypeptides, particulailv other polypeptides of animal origin

Fractionation and/or conventional purification methods can be used to obtain preparations of Ztryp3 purified fiom natural sources (e g testicular tissue), and recombinant Ztryp3 polypeptides and fusion Ztryp^ polypeptides purified from recombinant host cells In general, ammonium sulfate piecipitation and acid or chaotrope extraction may be used for fractionation of samples Exemplary purification steps may include hydroxyapatite size exclusion FPLC and reverse-phase high performance liquid chromatography Suitable chromatographic media include deπvatized dextrans, agarose, cellulose, poly aery lamide specialty silicas, and the like PEI, DEAE, QAE and Q derivatives are preferred Exemplary chromatographic media include those media deπvatized with phenyl, butyl, or octyl groups, such as Phenyl- Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas. Montgomeryv ille PA) Octyl-Sepharose (Pharmacia) and the like, or polyacryhc resins such as Amberchrom CG 71 (Toso Haas) and the like Suitable solid supports include glass beads, sihca- based resins, cellulosic resins, agarose beads cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used These supports may be modified with reactiv e groups that allow attachment of proteins by ammo groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties Examples of coupling chemistries include cyanogen bromide actu ation N-hvdroxysuccimmide activation, epoxide actu ation, sulfhydry l activation, hydiazide actu ation, and carboxyl and ammo derivativ es foi carbodnmide coupling chemistries These and other solid media aie well know n and widely used in the art and are available from commercial suppliers Selection of a particular method for polypeptide isolation and purification is a matter of routine design and is determined in part bv the properties of the chosen support See, for example Affinity Chtomatographx Pr inciples & Methods (Pharmacia LKB Biotechnology 1988). and Doonan. Pr otein Pur ification Pi otocols (The Humana Press 1996) Additional v anations in Ztryp3 isolation and puiitication can be dev ised by those of skill in the art Foi example, anti Ztryp antibodies obtained as described below can be used to isolate large quantities of protein by lmmunoaffimty purification

The polypeptides of the present inv ention can also be isolated bv exploitation of particular properties For example, immobilized metal ion adsorption (EM AC) chiomatography can be used to purify histidine-πch proteins, including those comprising polyhistidine tags Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem 3 1 (1985)) Histid e-πch proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (M Deutscher. (ed ). Meth Enzymol 182.529 ( 1990)) Within additional embodiments of the invention, a fusion of the polypeptide of interest and an affinitv tag (e g maltose-binding protein, an immunoglobuhn domain) may be constructed to facilitate purification Ztryp3 polypeptides or fragments thereof may also be piepared through chemical synthesis, as described above Ztryp3 polypeptides may be monomers or multimers, glycosylated or non-glycosylated, PEGylated or non-PEGylated. and may or may not include an initial methio ne amino acid residue

The present invention also contemplates chemically modified Ztιyp3 compositions, in which a Ztryp3 polypeptide is linked w ith a polymer Typically the polymer is water soluble so that the Ztryp3 conjugate does not precipitate in an aqueous environment, such as a physiological env ironment An example of a suitable poly mer is one that has been modified to have a single reactive group, such as an activ e ester for acylation. or an aldehyde for alkylation. In this way , the degree of polymerization can be controlled An example of a reactiv e aldehyde is polyethylene glycol propionaldehyde. or mono-(Cl-C lO) alkoxy . oi aryloxy derivatives thereof (see. for example. Harris, et al . U S Patent No 5.252 714) The polymer may be branched or unbranched Moreovei. a mixture of polymers can be used to produce Ztrvp3 conjugates

Ztryp3 coniugates used for therapy can comprise pharmaceutically acceptable watei -soluble polymer moieties Suitable w ater-soluble polymeis include polyethylene glycol (PEG) monomethoxv-PEG mono-(Cl -C10)alkoxy-PEG. aryloxv- PEG poly-(N-vιnyl pyrrohdone)PEG tresvl monomethoxy PEG PEG propionaldehyde. b/i-succimmidyl carbonate PEG. propvlene glycol homopolymers. a polypropylene oxide/ethylene oxide co-polymei polyoxyethylated polyols ( e g glycerol), polyvinyl alcohol, dextran. cellulose or other carbohydrate-based polymers Suitable PEG may hav e a molecular weight from about 600 to about 60.000 including for example. 5,000. 12.000. 20.000 and 25.000 A Ztryp3 conjugate can also comprise a mixture of such water-soluble polymers Antι-Ztιyp3 antibodies or anti ldiotvpe antibodies can also be conjugated with a watei -soluble polvmei

The present invention contemplates compositions comprising a peptide or polypeptide described herein Such compositions can further comprise a carrier The carrier can be a conventional organic or inorganic carπei Examples of carriers include water, buffer solution, alcohol, propvlene glycol. macrogol. sesame oil, corn oil,

Peptides and polypeptides of the present invention comprise at least six, at least nine, or at least 15 contiguous amino acid residues of an amino acid sequence comprising amino acid residues 20 to 235 of SEQ ID NO 2, or an amino acid sequence consisting of SEQ ID NO 2 Within certain embodiments of the invention the polypeptides comprise 20, 30, 40. 50, 100. or more contiguous residues of these am o acid sequences Additional polypeptides can compnse at least 15. at least 30. at least 40, or at least 50 contiguous ammo acids of amino acid residues 20 to 235 of SEQ ID NO.2 Nucleic acid molecules encoding such polypeptides are useful as polymerase chain reaction primers and probes

In addition to the uses described above, polynucleotides and polypeptides of the present invention are useful as educational tools in laboratory practicum kits for courses related to genetics and molecular biology, protein chemistry and antibody production and analysis Due to its unique polynucleotide and polypeptide sequences, molecules of Ztryp3 can be used as standards or as "unknow ns^' for testing purposes For example, Ztryp3 polynucleotides can be used as an aid. such as. for example, to teach a student how to prepaie expression constructs for bacterial. viral, or mammalian expression, including fusion constructs, wherein Ztryp3 is the gene to be expressed, for determining the restriction endonuclease cleavage sites of the polynucleotides determining mRNA and DNA localization of Ztryp3 polynucleotides in tissues (i.e.. by northern and Southern blotting as well as polymerase chain reaction), and for identifying related polynucleotides and polypeptides by nucleic acid hybridization As an illustration, students will find that Hhal digestion of a nucleic acid molecule consisting of the nucleotide sequence of nucleotides 42 to 746 of SEQ ID NO 1 provides fragments of about 284 base pairs, and 421 base pans, and that EcoRl digestion yields fragments of about 520 base pans, and 185 base pairs

Ztryp3 polypeptides can be used as an aid to teach preparation of antibodies; identifying proteins by western blotting, protein purification, determining the weight of expressed Ztryp3 polypeptides as a ratio to total protein expressed, identifying peptide cleavage sites, coupling amino and carboxy l terminal tags, amino acid sequence analysis, as well as, but not limited to monitoring biological activities of both the native and tagged protein (i e , protease inhibition) in

o Foi example, students will find that digestion of unglycosylated Ztryp s w ith hvdroxylamine yields three fragments naving approximate molecular weights of 6753 10397. and 9328. whereas digestion of unglycosylated Ztryp3 w ith mild acid hydrolysis yields fragments having approximate molecular weights of 2862. and 23598

Ztryp3 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, nucleai magnetic resonance spectroscopy to reveal the structure of proteins in solution. For example, a kit containing the Ztryp3 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 Ztryp3 would be unique unto itself

The antibodies which bind specifically to Ztryp3 can be used as a teaching aid to instruct students how to prepare affinity chromatography columns to purify Ztryp3, cloning and sequencing the polynucleotide that encodes an antibody and thus as a practicum for teaching a student how to design humanized antibodies The Ztrvp3 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 Ztr\p3 gene, polypeptide, or antibody are considered within the scope of the present invention 9. Production of Antibodies to Ztryp3 Proteins

Antibodies to Ztryp3 can be obtained, for example using as an antigen the product of a Ztryp3 expression vector or Ztryp3 isolated from a natural source Particularly useful antι-Ztryp3 antibodies "bind specifically with Ztryp3 Antibodies s are considered to be specifically binding if the antibodies exhibit at least one of the following two properties ( 1 ) antibodies bind to Ztryp3 with a threshold level of binding activity, and (2) antibodies do not significantly cross-react with polypeptides related to Ztryp3

With regard to the first characteristic, antibodies specifically bind if they 0 bind to a Ztryp3 polypeptide peptide or epitope w ith a binding affinity (K_d) of 10⁶ M ' or greater, pieferably 10 M ' oi greater, more preferably 10 M ' or greater and most pieferably 10 M ' or greatei The binding affinity of an antibody can be readilv determined by one of ordinal skill in the art foi example bv Scatchard analysis (Scatchard Ann NY Acad Sci 51 660 (1949)) With regard to the second 5 characteristic, antibodies do not significantly cross-react with related polypeptide molecules, for example, if they detect Ztryp3, but not know n related polypeptides using a standard Western blot analysis Examples of known related polypeptides are orthologs and proteins from the same species that are membeis of a protein family For example, specifically-binding antι-Ztryp3 antibodies bind with Ztryp3, but not with 0 known serine proteases, such as trypsin. tryptase. kalhkrein. chymotrypsin, subtihsin, prostate specific antigen, chymotryptic enzyme of skin, and protease M. and the like

Antι-Ztryp3 antibodies can be pioduced using antigemc Ztryp3 epitope- beaπng peptides and polypeptides Antigemc epitope-beaπng peptides and polypeptides of the present inv ention contain a sequence of at least nine, or between 15 5 to about 30 amino acids contained within SEQ ID NO 2 However, peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that bind with Ztryp3 It is desirable that the amino acid sequence of the 0 epitope-beaπng peptide is selected to provide substantial solubility in aqueous solv ents (i e , the sequence includes relatively hydrophihc residues while hvdrophobic residues are preferably avoided) Moreover, ammo acid sequences containing prohne residues may be also be desirable for antibody production

As an illustration, potential antigemc sites in Ztryp3 were identified using the Jameson-Wolf method, Jameson and Wolf. CABIOS 4 181, (1988) as implemented by the PROTEAN program (version 3 14) of LASERGENE (DNASTAR, Madison, WI) Default parameters were used in this analysis The Jameson-Wolf method predicts potential antigemc determinants by combining six major subroutines for protein structural prediction Briefly, the Hopp- Woods method, Hopp et al, Proc. Nat'l Acad Sci USA 78 3824 (1981 ), was first used to identify ammo acid sequences representing areas of greatest local hydrophilicity (parameter, seven residues averaged) In the second step. Emi 's method. Emini et al J Virology 55:836 ( 1985), was used to calculate surface probabilities (parameter surface decision threshold (0.6) = 1 ) Third, the Karplus-Schultz method. Karplus and Schultz. Naturwissenschaften 72.212 (1985). w as used to predict backbone chain flexibility (parameter: flexibility threshold (0 2) = 1 ) In the fourth and fifth steps of the analysis, secondaiy stmcture predictions weie applied to the data using the methods of Chou-Fasman. Chou. "Prediction of Protein Stiuctuial Classes from Amino Acid Composition.^" in Pr ediction of Protein Str uctur e and the Principles of Pr otein Conformation. Fasman (ed ). pages 549-586 (Plenum Press 1990). and Gamier-Robson Gamier et al , J Mol Biol 120 97 (1978) (Chou-Fasman parameters conformation table = 64 proteins, α region threshold = 103, β region threshold = 105, Garmei- Robson parameters' and β decision constants = 0) In the sixth subroutine, flexibility parameters and hydropathy/solvent accessibility factois were combined to determine a surface contour value, designated as the "antigemc index " Finally, a peak broadening function was applied to the antige c index, which broadens major surface peaks by adding 20. 40, 60, or 807c of the respecti e peak value to account foi additional free energy derived from the mobility of surface regions relative to interior regions This calculation was not applied, however, to any major peak that resides in a helical region, since helical regions tend to be less flexible

The results of this analysis indicated that the following amino acid sequences of SEQ ID NO.2 would provide suitable antigemc molecules ammo acid residues 18 to 28 ("antigemc molecule 1"). ammo acid residues 68 to 78 ("antigemc molecule 2"), amino acid residues 95 to 100 ("antigemc molecule 3"), amino acid residues 124 to 131 ("antigemc molecule 4"). ammo acid residues 138 to 154 ("antigemc molecule 5"), and amino acid lesidues 160 to 176 ("antige c molecule 6") The present invention contemplates the use of any one of antigemc molecules 1 to 6 to generate antibodies to Ztryp3 The present invention also contemplates polypeptides comprising at least one of antigemc molecules 1 to 6

Polvclonal antibodies to recombinant Ztryp3 protein or to Ztryp3 isolated from natural sources can be prepared using methods well-known to those of skill in the art. Antibodies can also be generated using a Ztryp3-glutathιone transferase fusion protein, which is similar to a method descπbed by Burrus and McMahon, Exp Cell. Res 220:363 (1995) General methods for pioducing polvclonal antibodies are described, for example, by Green et al "Production of Polvclonal Antisera.^" in Immunochemical Protocols (Manson. ed ), pages 1-5 (Humana Press 1992). and Williams et al . "Expression of foreign proteins in E coli using plasmid v ectois and purification of specific polvclonal antibodies.^" in DNA Cloning 2 Expiession Sy stems, 2nd Edition. Glover et al (eds ). page 15 (Oxford university Press 1995)

The lmmunogemcity of a Ztryp3 polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund s complete oi incomplete adjuvant Polypeptides useful for immunization also include fusion polypeptides. such as fusions of Ztryp3 or a portion thereof with an immunoglobuhn 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-hke.^" such portion may be adv antageously joined or linked to a maciomolecular carπei (such as keyhole limpet hemocyanin (KLH). bovine serum albumin (BS A ) or tetanus toxoid) foi immunization Although polyclonal antibodies are typically raised in animals such as horse, cow. dog. chicken, rat. mouse, rabbit, goat, guinea pig. oi sheep, an antι-Ztryp3 antibody of the present invention may also be derived from a subhuman primate antibody General techniques for raising diagnostically and therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al . international patent publication No WO 91/1 1465, and in Losman et al hit J Cancel 46 310 (1990)

Alternatively , monoclonal antι-Ztryp3 antibodies can be generated Rodent monoclonal antibodies to specific antigens may be obtained by methods kno n to those skilled in the ait (see. for example. Kohlei et al Natiti e 256 495 ( 1975 ). Cohgan et al (eds ), Current Protocols in Immunology . Vol 1. pages 2 5 1-2 6 7 (John Wiley & Sons 1991) ["'Cohgan"], Picksley et al "Production of monoclonal antibodies against proteins expressed in E coli," in DNA Cloning 2 Expression Systems. 2nd Edition. Glover et al (eds ), page 93 (Oxford University Press 1995))

Briefly, monoclonal antibodies can be obtained by meeting mice w ith a composition comprising a Ztrxp3 gene product, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-l mphocytes, fusing the B-lymphocytes with myeloma cells to produce hybπdomas cloning the hybπdomas, selecting positive clones which produce antibodies to the antigen, cultuπng the clones that produce antibodies to the antigen, and isolating the antibodies from the hybπdoma cultures

In addition, an antι-Ztryp3 antibody of the present invention may be derived from a human monoclonal antibody Human monoclonal antibodies are obtained from transge c mice that hav e been engineered to produce specific human antibodies in response to antigemc challenge In this technique elements of the human heav y and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci The transgemc mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hvbπdomas Methods foi obtaining human antibodies from transgemc mice are described for example, by Green et al Native Genet 7 13 (1994). Lonberg et al Nature 368 856 (1994), and Taylor et al hit Immun 6 579 (1994) Monoclonal antibodies can be isolated and purified from hybtidoma cultures bv a variety of well-established techniques Such isolation techniques include affinity chromatography ith Protein A Sepharose size-exclusion chromatography and ion exchange chromatography (see foi example Cohgan at pages 2 7 1-2 7 12 and pages 2 9 1-2 9 3 Barnes et al "Purification of Immunoglobuhn G (IgG) in Methods in Molecular Biology , Vol 10, pages 79-104 (The Humana Press, Inc 1992))

For particular uses it may be desirable to prepare fragments of anti- Ztryp3 antibodies Such antibody fragments can be obtained, for example by proteolytic hydrolysis of the antibody Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods As an illustration, antibody fragments can be produced by enzvmatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab )-> This fragment can be further cleaved using a thiol reducing agent to produce 3 5S Fab monovalent fragments Optionally the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages As an alternative, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly These methods aie described for example, by Goldenberg, U S patent No 4,331,647. Nisonoff et α/ Arch Biochem Biophy s 59 230 (1960) Porter. Biochem J 73 1 19 (1959), Edelman et al , in Methods in Enzy ologx Vol 1. page 422 (Academic Press 1967). and by Cohgan at pages 2 8 1-2 8 10 and 2 10 2 10 4 Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used so long as the fragments bind to the antigen that is recognized by the intact antibody

For example Fv fragments comprise an association of NH and N chains This association can be noncovalent. as described by Inbar et al Proc Nat I Acad Sci USA 69 2659 (1972) Alternatively the variable chains can be linked bv an mtermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde (see. foi example. Sandhu. Cut Re \ Biotech 12 437 ( 1992))

The Fv fragments may comprise V_H and V chains, which are connected by a peptide linker These single-chain antigen binding proteins (scFv ) are prepared by constructing a structural gene comprising DNA sequences encoding the V_H and V_L domains which are connected by an ohgonucleotide The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell, such as E coli The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains Methods for producing scFv s aie described, foi example, by Whitlow et al Methods 4 Companion to Methods n Enzxnwlogx 2 97 ( 1991 ) (also see. Bird et al Science 242 423 ( 1988 ) Ladner et al L S Patent No 4.946.778. Pack et al . Bio/Technology 11 1271 ( 1993 ). and Sandhu. supra )

As an illustration, a scF\ can be obtained bv exposing ly mphocy tes to Ztryp3 polypeptide in vitr o, and selecting antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled Ztryp3 protein or peptide) Genes encoding polypeptides having potential Ztryp3 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis These random peptide display libraries can be used to screen for peptides. which interact with a know n target which can be a protein or polypeptide. such as a ligand or receptor, a biological or synthetic macromolecule. or organic or inorganic substances Techniques for creating and scieemng such random peptide display libraries are known in the art (Ladner et al U S Patent No 5.223.409. Ladner et al , U.S. Patent No 4,946.778. Ladner et al , U S Patent No 5.403.484, Ladner et al, U.S Patent No 5.571 ,698. and Kay et al . Phage Display of Peptides and Proteins (Academic Press, Inc 1996)) and random peptide display libraries and kits for screening such libraries are available commercially , for instance from CLONTΕCH Laboratories. Inc (Palo Alto. CA). Invitrogen Inc (San Diego. CA), New England Biolabs, Inc (Beverly. MA), and Pharmacia LKB Biotechnology Inc (Piscataway. NJ) Random peptide display libraries can be screened using the Ztryp3 sequences disclosed herein to identify proteins, which bind to Ztryp3

Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR) CDR peptides ("minimal recognition units^") can be obtained by constructing genes encoding the CDR of an antibody of interest Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the v ariable region from RNA of antibody -producing cells (see. for example Larπck et al Methods 4 Companion to Methods in Enzxmology 2 106 ( 1991 ) Courtenav -Luck "Genetic Manipulation ot Monoclonal Antibodies in Monoclonal Antibodies Production Engineer ing and Clinical Application. Ritter et al (eds ), page 166 (Cambridge University Press 1995 ) and W aid et al ' Genetic Manipulation and Expiession of Antibodies ^" in Monoclonal Antibodies Pi maples and Applications. Birch et al (eds ), page 137 (Wiley -Liss Inc 1995))

Alternatively , an antι-Ztryp3 antibodv may be derived from a "humanized" monoclonal antibody Humanized monoclonal antibodies are produced bv transferring mouse complementary determining iegions from heav y and light variable chains of the mouse immunoglobuhn into a human variable domain Tvpical residues of human antibodies are then substituted in the framew oik regions ot the muπne counterparts The use of antibody components deriv ed from humanized monoclonal antibodies obviates potential problems associated w ith the irnmunogemcitv of muπne constant regions General techniques for cloning muπne immunoglobuhn variable domains are described for example by Orlandi et al Pi oc Nat 1 Acad Sa USA 86 3833 ( 1989) Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al Nature 321 522 ( 1986). Carter et al Proc ΛαtV Acad Sa USA 59 4285 ( 1992). Sandhu. Crit Re\ Biotech 12 437 ( 1992). Singer et al . J Imrnun 150 2844 ( 1993). Sudhir (ed ), Antibody Engineerin g Protocols (Humana Press, Inc 1995), Kelley "Engineering Theiapeutic Antibodies," in Pr otein Engineer ing Principles and Pi actice. Cleland et al (eds ) pages 399-434 (John \\ lley & Sons. Inc 1996). and by Queen et al , U S Patent No 5 693,762 ( 1997 )

Polvclonal anti-idiotype antibodies can be prepared by immunizing animals with antι-Ztryp3 antibodies or antibody fragments, using standard techniques See, for example, Green et al . "Production of Polvclonal Antisera," in Methods In Molecular Biology Immunochermcal Protocols, Manson (ed ). pages 1 -12 (Humana Press 1992) Also, see Cohgan at pages 2 4 1 -2 4 7 Alternatively , monoclonal anti- ldiotype antibodies can be prepared using antι-Ztryp3 antibodies or antibody fragments as immunogens with the techniques, described abov e As another alternativ e humanized anti-idiotype antibodies or subhuman primate anti-idiotype antibodies can be prepared using the above-described techniques Methods for producing anti-idiotvpe antibodies are described, for example, by Iπe. U S Patent No 5.208.146, Greene et al , U.S Patent No 5.637.677. and Varthakav i and Minocha. J Gen Virol 77 1875 (1996) Anti-idiotype Ztryp3 antibodies, as well as Ztryp3 polypeptides can be used to identify and to isolate Ztryp3 substiates and inhibitoi s For example, proteins and peptides of the present inv ention can be immobilized on a column and used to bind substrate and inhibitor proteins from biological samples that are run over the column (Hermanson et al (eds ) Immobilized Affinity Ligand Techniques, pages 195-202 (Academic Press 1992)) Radiolabeled or affinity labeled Ztryp3 polypeptides can also be used to identify or to localize Ztryp3 substrates and inhibitors in a biological sample (see foi example, Deutscher (ed ) Methods in Enzxmol , vol 182 pages 721 -37 (Academic Press 1990). Brunner et α/ Ann

Biochem 62 483 ( 1993 ) Fedan et al Biochem Pharmacol 33 1 167 ( 1984))

10. Use ofZtryp3 Nucleotide Sequences to Detect Ztryp3 Gene Expression and to Examine Ztryp3 Gene Structure

Nucleic acid molecules can be used to detect the expression of a Ztrvp3 gene in a biological sample Such probe molecules include double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO 1 oi a portion 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 ith regions of a Ztι yp3 gene that hav e a low sequence similarity to comparable regions in other serine proteases In a basic assay a single-stianded probe molecule is incubated w ith

RNA. isolated from a biological sample, under conditions of temperatuie and ionic strength that promote base pairing between the probe and target Ztryp3 RNA species After separating unbound probe from hvbiidized molecules the amount of hvbπds is detected 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 αl (eds ) "Analysis of Gene Expression at the RNA Level," in Methods Gene Biotechnology pages 225 239 (CRC Press. Inc 1997)) Nucleic acid probes can be detectably labeled with radioisotopes such as ^"P or S Alternatively, Ztryp3 RNA can be detected with a noniadioacuve hybridization method (see. for example. Isaac (ed ). Protocols foi Nucleic Acid Analysis by oniadtoactn e Probes (Humana Press, Inc 1993)) Typically noniadioacuve detection is acnieved by enzymatic conversion of chromogenic oi chemilummescent substrates Illustiativ e nonradioactive moieties include biotin, fluorescein. and digoxigemn Ztryp3 ohgonucleotide probes are also useful foi in

o diagnosis As an illustration ' F-labeled oligonucleotides can be admimsteied to a sub]ect and visualized by positron emission tomography (Tavitian et al Natitie Medicine 4 467 ( 1998))

Numerous diagnostic procedures take advantage ot tnε polvmerase chain reaction (PCR) to increase sensitiv ity of detection methods Standard techniques for performing PCR are well-known (see, generally . Mathew (ed ) Piotocols in Human Moleculai Genetics (Humana Press Inc 1991 ). White (ed ) PCR Pi otocols Cur rent Methods and Applications (Humana Press, Inc 1993) Cotter (ed j Moleculai Diagnosis of Cancel (Humana Press. Inc 1996). Hanausek and \\ alaszek (eds ), Tumor Marker Pr otocols (Humana Piess Inc 1998). Lo (ed ) Clinical Applications of PCR (Humana Press, inc 1998). and Meltzer (ed ) PCR in Bioana sis (Humana Piess. Inc 1998))

PCR primers can be designed to amplify a portion ot the Ztr xp3 gene that has a low sequence similarity to a comparable region in other serine proteases One variation of PCR for diagnostic assays is reverse transcπptase-PCR

(RT-PCR) In the RT-PCR technique. RNA is isolated from a biological sample, reveise transcribed to cDNA, and the cDNA is incubated with Ztr p3 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 aie analyzed using standard techniques

As an illustration RNA is isolated from biological sample using, tor example, the guamdinium-thiocvanate cell lysis procedure described abov e Alternatively, a solid-phase technique can be used to isolate mRN A from a cell lysate A reverse transcription reaction can be primed with the isolated RNA using random oligonucleotides, short homopolymers of dT, or Ztryp3 anti-sense o gomers Ohgo-dT primers offer the ad antage that v arious mRNA nucleotide sequences are amplified that can provide control target sequences Ztryp3 sequences are amplified by the polymerase chain reaction using tw o flanking ohgonucleotide primers that are typically 20 bases in length 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 detectabl -labeled Ztryp3 probe, and examined by autoradiography Additional alternative approaches include the use of digoxigenin-labeled deoxyπbonucleic acid tπphosphates to provide chemiluminescence detection, and the C-TRAK coloπmetπc assav Another approach for detection of Ztryp3 expression is cycling probe technology (CPT). in which a single-stranded DNA target binds with an excess of DNA-RNA-DNA chimeπc probe to form a complex, the RNA portion is cleaved w ith RNAase H. and the presence of cleaved chimeπc 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 Ztryp3 sequences can utilize approaches such as nucleic acid sequence-based amplification (NASBA). cooperative amplification of templates by cross-hybridization (CATCH ), and the hgase chain reaction (LCR) (see for example. Marshall et al . U.S Patent No 5.686.272 ( 1997). Dyer et al J Vnol Methods 60 161 ( 1996). Ehπcht et al, Em J. Biochem 243 358 ( 1997). and Chadw ick et al. J Vn ol Methods 70 59 ( 1998)) Other standai d methods are know n to those ot skill in the ait

Ztrvp3 probes and pπmei s can also be used to detect and to localize Ztryp3 gene expression in tissue samples Methods for such situ hybridization aie well-known to those of skill in the art (see. foi 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 w ell-known to those ol skill in the art (see. foi example. Mathew (ed ). Pi otocols Human Moleculai Genetics (Humana Press. Inc 1991 ). Coleman and Tsongahs. Moleculai Diagnostics (Humana Press. Inc 1996). and Elles. Moleculai Diagnosi s of Genetic Diseases (Humana Press, Inc., 1996)).

Nucleic acid molecules comprising Ztryp3 nucleotide sequences can be used to determine whether a subject's chromosomes contain a mutation in the Ztryp3 gene Detectable chromosomal aberrations at the Ztryp3 gene locus include, but aie not limited to. aneuploidy. gene copy number changes, insertions, deletions, restriction site changes and rearrangements Of particular interest aie genetic alterations that inactiv ate a Ztrχp3 gene

Aberrations associated with a Ztryp3 locus can be detected using nucleic acid molecules of the piesent 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 Tsongahs, Molecular Diagnostics (Human Press. Inc 1996) Elles (ed ) Moleculai Diagnosis of Genetic Diseases (Humana Press Inc 1996). Landegren (ed ) Laborator y 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 W lley & Sons 1998). and Richards and Ward, "Molecular Diagnostic Testing in Principles of Molecular Medicine, pages 83-88 (Humana Press. Inc 1998)) The protein truncation test is also useful for detecting the lnactuation 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 DN A PCR is then used to amplify the Ztryp3 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 RN A 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 Genetic s pages 9 1 1 1 - 9 1 1 18 (John Wiley & Sons 1998)

The chromosomal location of the Ztι xp3 gene can be determined using radiation hybrid mapping, which is a somatic cell genetic technique developed for constructing high-resolution, contiguous maps ot mammalian chromosomes (Cox et al Science 250 245 (1990)) Partial or full know ledge of a gene's sequence allows one to design PCR primers suitable for use with chromosomal radiation hybrid mapping panels Radiation hybrid mapping panels are commercially available which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBπdge 4 RH Panel (Research Genetics, Inc , Huntsville. AL) These panels enable rapid PCR-based chromosomal localizations and ordering of genes, sequence-tagged sites, and other nonpolymorphic and polymorphic markers within a region of interest This includes establishing direcfly proportional physical distances bet een newly discovered genes of interest and previously mapped markers

The present invention also contemplates kits for performing a diagnostic assay for Ztr yp3 gene expression or to analyze the Ztryp3 locus of a subject Such kits comprise nucleic acid probes such as double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO 1 or a portion 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 Illustrativ e portions reside ithin nucleotides 42 to 746 of SEQ ID NO 1 , or within nucleotides 99 to 746 of SEQ ID NO 1 Probe molecules may be DNA, RNA. oligonucleotides. and the like Kits may comprise nucleic acid primers for performing PCR

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 Ztryp3 probe or primer The kit mav also comprise a second container comprising one or moie reagents capable of indicating the presence of Ztryp3 sequences Examples of such indicator reagents include detectable labels sucn as radioactiv e labels, fluoiochromes. chemiluminescent agents, and the like A kit may also compnse a means for conveying to the user that the Ztιyp3 probes and primers are used to detect Ztryp3 gene expression For example ritten instructions mav state that the enclosed nucleic acid molecules can be used to detect either a nucleic acid molecule that encodes Ztryp3, or a nucleic acid molecule having a nucleotide sequence that is complementary to a Ztryp3-encodmg nucleotide sequence or to analyze chromosomal sequences associated with the Ztryp3 locus The written material can be applied directly to a container, or the written material can be provided in the form ot a packaging insert

11. Use of Anti-Ztryp3 Antibodies to Detect Ztryp3 Protein

The present invention contemplates the use of antι-Ztryp3 antibodies to screen biological samples in vitro foi the presence of Ztryp3 In one type of in \ ιtιo assay . antι-Ztryp3 antibodies are used in liquid phase For example, the piesence of Ztryp3 in a biological sample can be tested by mixing the biological sample with a trace amount of labeled Ztryp3 and an antι-Ztryp3 antibody under conditions that promote binding between Ztryp3 and its antibody Complexes of Ztryp3 and antι-Ztryp3 in the sample can be separated from the reaction mixture by contacting the complex with an immobilized protein hich binds with the antibody, such as an Fc antibody or Staphylococcus protein A The concentration of Ztryp3 in the biological sample will be inversely proportional to the amount of labeled Ztryp3 bound to the antibody and directly related to the amount of free labeled Ztryp3

Alternatively, in

assays can be performed in which antι-Ztryp3 antibody is bound to a solid-phase earner For example, antibody can be attached to a polymer, such as aminodextian, in order to link the antibody to an insoluble support such as a polymer-coated bead, a plate or a tube Other suitable in

assays will be readily apparent to those of skill in the art 6^

In another approach, antι-Ztryp3 antibodies can be used to detect Ztryp3 in tissue sections piepared from a biopsy specimen Such lmmunochemical detection can be used to determine the relative abundance of Ztιyp3 and to determine the distribution of Ztryp3 in the examined tissue General lmmunochemistry techniques are well established (see. foi example. Ponder, "Cell Marking Techniques and Their Application ^' in Mammalian Development A Practical Approach. Monk (ed ), pages 1 15-38 (IRL Press 1987). Cohgan at pages 5 8 1-5 8 8, Ausubel ( 1995) at pages 14 6 1 to 14 6 13 (Wiley Interscience 1990), and Manson (ed ) Methods In Moleculai Biology Vol 10 lmmunochemical Piotocols (The Humana Piess. Inc 1992)) lmmunochemical detection can be peitoπned by contacting a biological sample with an anϋ-Ztryp3 antibody, and then contacting the biological sample w ith a detectablv labeled molecule which binds to the antibody For example the detectablv labeled molecule can comprise an antibodv moietv that binds to antι-Ztrvp antibodv Alternatively, the antι-Ztryp3 antibody can be conjugated with avidin/streptav idin (or biotin) and the detectably labeled molecule can comprise biotin (or avidin/streptavidin) Numerous variations of this basic technique aie well-known to those of skill in the art

Alternatively, an antι-Ztryp3 antibody can be conjugated with a detectable label to form an antι-Ztryp3 immunoconjugate Suitable detectable labels include, for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label or colloidal gold Methods of making and detecting such detectably- labeled lmmunoconjugates are well-know n to those of ordinaiy skill in the art. and are described in more detail below

The detectable label can be a ladioisotope that is detected by autoradiography Isotopes that are particularly useful for the purpose of the present invention are \H, ^{X2 π ι}I, "S and ¹⁴C

Antι-Ztryp3 immunoconjugate s can also be labeled with a fluorescent compound The presence of a fluorescently-labeled antibody is determined by exposing the immunoconjugate to light of the proper wavelength and detecting the resultant fluorescence Fluorescent labeling compounds include fluorescein isothiocyanate. lhoda- mine, phycoerytheπn, phycocyanin, allophycocyamn, o-phthaldehyde and fluorescamine

Alternatively. antι-Ztryp3 lmmunoconjugates can be detectably labeled by coupling an antibody component to a chemiluminescent compound The presence of the chemiluminescent-tagged immunoconjugate is determined by detecting the presence of luminescence that arises during the course of a chemical reaction Examples of chemi- luminescent labeling compounds include luminol, isoluminol, an aromatic acπdinium ester, an lmidazole, an acπdinium salt and an oxalate estei Similarly, a biolurmnescent compound can be used to label antι-Ztryp3 lmmunoconjugates of the present invention Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction The presence of a biolurmnescent protein is determined by detecting the piesence of luminescence Biolurmnescent compounds that are useful for labeling include luciferm. luciferase and aequoπn

Alternatively. antι-Ztryp3 lmmunoconjugates can be detectably labeled by linking an antι-Ztryp3 antibody component to an enzyme When the antι-Ztryp3 enzyme conjugate is incubated in the presence of the appropriate substrate the enzyme moiety reacts with the substrate to produce a chemical moietv which can be detected, foi example, by spectrophotometπc. fluorometπc oi v isual means Examples of enzymes that can be used to detectably label polyspecific ιmmunocon)ugates include β-galactosidase glucose oxidase, peroxidase and alkaline phosphatase

Those of skill in the art will know of other suitable labels which can be employed in accordance with the present invention The binding ot marker moieties to antι-Ztryp3 antibodies can be accomplished using standard techniques known to the art Typical methodology in this regard is described by Kennedy et al Clin Chun Ada 70 1 (1976), Schurs et al , Clin Chun Ada 81 1 (1977), Shih et al Int I J Cancel 46 1 101 (1990), Stein et al , Cancer Res 50 1330 (1990). and Cohgan, supra Moreover, the convenience and versatility of lmmunochemical detection can be enhanced by using antι-Ztryp3 antibodies that hav e been conjugated with avidin, streptavidin. and biotin (see, for example, Wilchek et al (eds ), "Avidin-Biotin Technology." Methods In Enzymology Vol 184 (Academic Press 1990), and Bayer et al "lmmunochemical Applications of Avidin-Biotin Technology .^" in Methods In Moleculai Biology, Vol 10, Manson (ed ). pages 149-162 (The Humana Press. Inc 1992)

Methods for performing immunoassays are well-established See, for example, Cook and Self, "Monoclonal Antibodies m Diagnostic Imrnunoassays,^" in Monoclonal Antibodies Production, Engineering and Clinical Application. Ritter and Ladyman (eds ), pages 180-208. (Cambridge University Press, 1995) Perry, "The Role of Monoclonal Antibodies in the Advancement of Immunoassav Technology.^" in Monoclonal Antibodies Principles and Applications. Birch and Lennox (eds ), pages 107-120 (Wiley-Liss, Inc 1995), and Diamandis, hnmunoassay ( Academic Press, Inc 1996)

In a related approach, biotin- or FLTC-labeled Ztryp3 can be used to identify cells that bind Ztryp3 Such can binding can be detected, for example, using flow cytometry The present invention also contemplates kits for performing an lmmunological diagnostic assay foi Ztrχp3 gene expression Such kits comprise at least one container comprising an antι-Ztryp3 antibody, or antibody fragment A kit may also comprise a second container comprising one or more reagents capable of indicating the presence of Ztryp3 antibody or antibody fragments Examples of such indicatoi reagents include detectable labels such as a radioactiv e label, a fluorescent label, a chemiluminescent label, an enzyme label, a biolurmnescent label, colloidal gold and the like A kit may also comprise a means for conveying to the user that Ztryp3 antibodies oi antibody fragments are used to detect Ztryp3 protein For example written instructions mav state that the enclosed antibody or antibodv ftagment can be used to detect Ztryp3 The written mateπal can be applied directly to a container, or the πtten material can be provided in the form of a packaging insert

12. Therapeutic Uses of Polypeptides Having Ztryp3 Activity The present invention includes the use of proteins, polypeptides, and peptides having Ztryp3 activity (such as Ztryp3 polypeptides. anti-idiotype antι-Ztryp3 antibodies, and Ztryp3 fusion proteins) to a subject who lacks an adequate amount of this seπne protease

In general, Ztryp3 agonists and antagonists can be useful for treatment of disorders associated with asthma, the immune system, gastiointestinal system heart inflammation, lymph system, and testis The polypeptides, agonists, antagonists, nucleic acid, and antibodies of the present invention can be used in treatment of disorders associated with asthma vascular function such as stroke, inflammation, and testicular function As an illustration, the tissue specificity of Ztryp3 expression suggests a role in spermatogenesis, indicating that Ztryp3 polypeptides, and its agonists may prove useful in modulating spermatogenesis

Generally, the dosage of administered polypeptide, protein or peptide will vary depending upon such factors as the subject s age weight, height, sex, general medical condition and previous medical history Typically , it is desirable to provide the recipient with a dosage of a molecule having Ztryp3 activity, which is in the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of subject), although a lower or higher dosage also may be administered as circumstances dictate

Administration of a molecule having Ztryp3 activity to a subiect can be intravenous, intraarterial, intrapeπtoneal, intramuscular, subcutaneous, intrapleural lntrathecal. by perfusion through a regional catheter, or by direct intralesional injection When administering therapeutic proteins by injection, the administration may be by continuous infusion or by single or multiple boluses.

A pharmaceutical composition comprising a protein, polypeptide. or peptide having Ztryp3 activity can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic proteins are combined in a mixture with a pharmaceutically acceptable carrier. A composition is said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well-known to those in the art. See. for example. Gennaro (ed. ). Remington's Pharmaceutical Sciences. 19th Edition (Mack Publishing Company 1995 ).

For purposes of therapy, molecules having Ztryp3 activity and a pharmaceutically acceptable carrier are administered to a patient in a therapeutically effective amount. A combination of a protein, polypeptide. or peptide having Ztryp3 activity and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.

A pharmaceutical composition comprising molecules having Ztryp3 activity can be furnished in liquid form, or in solid form. Liquid forms, including liposome-encapsulated formulations, are illustrated by injectable solutions and oral suspensions. Exemplary solid forms include capsules, tablets, and controlled-release forms, such as a miniosmotic pump or an implant. Other dosage forms can be devised by those skilled in the art. as shown, for example, by Ansel and Popovich. Pharmaceutical Dosage Forms and Drug Delivery Systems, 5' Edition (Lea & Febiger 1990), Gennaro (ed.), Remington 's Pharmaceutical Sciences, 19' Edition (Mack Publishing Company 1995). and by Ranade and Hollinger, Drug Deliver-y Systems (CRC Press 1996).

As an illustration, Ztryp3 pharmaceutical compositions may be supplied as a kit comprising a container that comprises Ztryp3. Ztryp3 can be provided in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection. Such a kit may further comprise written information on indications and usage of the pharmaceutical composition. Moreover, such information may include a statement that the Ztryp3 composition is contraindicated in patients with known hypersensitivity to Ztryp3. 13. Therapeutic Uses of Ztryp3 Nucleotide Sequences

The present invention includes the use of Ztryp3 nucleotide sequences to provide Ztryp3 to a subject in need of such treatment In addition, a therapeutic expiession vector can be provided that inhibits Ztryp3 gene expression such as an anti- sense molecule, a πbozyme. or an external guide sequence molecule

There are numerous approaches to introduce a Ztryp3 gene IO a sub)ect including the use of recombinant host cells that express Ztryp3. de verv of naked nucleic acid encoding Ztryp3, use of a catiomc hpid carπei with a nucleic acid molecule that encodes Ztryp3. and the use of viruses that express Ztryp3. such as recombinant retroviruses recombinant adeno-associated viruses recombinant adenoviruses, and recombinant Herpes simplex viruses (see for example Mulligan Science 260926 (1993). Rosenbeig et αl Science 24215⁷5 (1988) LaSalle et αl Science 259988 (1993), Wolff et αl Science 24⁷1465 (1990). Bieaklield and Deluca The New Biologist 3203 (1991)) In an ex

approach, for example cells are isolated from a subject, transfected with a vector that expresses a Ztιyp3 gene, and then transplanted into the subject

In order to effect expression of a Ztrxp3 gene, an expression vector is constructed in which a nucleotide sequence encoding a Ztryp3 gene is operably linked to a core promoter, and optionally a regulatory element, to control gene transcription The general requirements of an expression vector aie described above

Alternatively, a Ztryp3 gene can be delivered using lecombinant viral vectors, including for example, adenoviral vectois (e g . Kass-Eisler et αl Proc Nat I Acad Sa USA 9011498 (1993) Kolls et al Proc Nat I Acad Sa USA 91215 (1994), Li et al Hum Gene Ther 4403 (1993), Vincent et al hat Genet 5130 (1993), and Zabner et al Cell 75.207 (1993)), adenovirus-associated viral vectors (Flotte et al, Proc Nat'l Acad Sa USA 9010613 (1993)). alphaviruses such as Semhki Forest Virus and Sindbis Virus (Hertz and Huang. J Vit 66857 (1992), Raju and Huang, J Vir 65.2501 (1991). and Xiong etal. Science 2431188 (1989)). herpes viral vectors (eg. US Patent Nos 4.769.331.4.859.587.5.288.641 and 5.328.688). parvovirus vectors (Koeπng et al Hum Gene Therap 5457 (1994)). pox virus vectors (Ozaki et al , Biochem Biophys Res Cornrn 193653 (1993). Panicah and Paoletti. Proc Nat'l Acad Sa USA 794927 (1982)), pox viruses such as canary pox virus or vaccinia virus (Fisher-Hoch etal Proc Nat'l Acad Sa USA 56317 (1989), and Flexneretα/ Arm N Y Acad Sa 56986 (1989)). and retroviruses (e g, Baba et al , J Neurosurg 79729 (1993). Ram et al , Cancer Res 5383 (1993). Takamiya et al , J Neuros Res 33493 (1992) Vile and Hart. Cancer Res 53962 (1993). Vile and Hart. Cancer Res 553860(1993) and Anderson et al US Patent No 5.399.346) Within various embodiments, either the viral vector itself, or a viral particle which contains the viral vector may be utilized in the methods and compositions described below.

As an illustration of one system, adenovirus. a double-stranded DNA virus, is a well-characterized gene transfer vector for delivery of a heterologous nucleic acid molecule (for a review, see Becker et al. Meth. Cell Biol 43: 161 ( 1994): Douglas and Curiel. Science & Medicine 4:44 (1997 )). The adenovirus system offers several advantages including: (i) the ability to accommodate relatively large DNA inserts, (ii ) the ability to be grown to high-titεr, (iii ) the ability to infect a broad range of mammalian cell types, and (iv) the ability to be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. In addition, adenoviruses can be administered by intravenous injection, because the viruses are stable in the bloodstream.

Using adenovirus vectors where portions of the adenovirus genome are deleted, inserts are incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. In an exemplary system, the essential El gene is deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell. When intravenously administered to intact animals, adenovirus primarily targets the liver. Although an adenoviral delivery system with an El gene deletion cannot replicate in the host cells, the host's tissue will express and process an encoded heterologous protein. Host cells will also secrete the heterologous protein if the corresponding gene includes a secretory signal sequence. Secreted proteins will enter the circulation from tissue that expresses the heterologous gene (e.g., the highly vascularized liver).

Moreover, adenoviral vectors containing various deletions of viral genes can be used to reduce or eliminate immune responses to the vector. Such adenoviruses are El -deleted, and in addition, contain deletions of E2A or E4 (Lusky et al, J. Virol. 72:2022 (1998); Raper et al. Human Gene Therapy 9:671 ( 1998)). The deletion of E2b has also been reported to reduce immune responses (Amalfitano et al, J. Virol. 72:926 (1998)). By deleting the entire adenovirus genome, very large inserts of heterologous DNA can be accommodated. Generation of so called "gutless" adenoviruses. where all viral genes are deleted, are particularly advantageous for insertion of large inserts of heterologous DNA (for a review, see Yeh. and Perricaudet. FASEB J. 11:615 (1997)).

High titer stocks of recombinant viruses capable of expressing a therapeutic gene can be obtained from infected mammalian cells using standard methods. For example, recombinant HSV can be prepared in Vero cells, as described by Brandt et al. J. Gen. Virol. 72:2043 ( 1991 ). Herold et al, J. Gen. Virol. 75: 121 1 (1994). Visalli and Brandt. Virology 185:419 ( 1991 ). Grau et al, Invest. Ophthalmol. Vis. Sa. 30:2474 (1989). Brandt et al, J. Virol Meth. 36:209 ( 1992). and by Brown and MacLean (eds. ). HSV Virus Protocols (Humana Press 1997 )

Alternatively, an expression vector comprising a Ztryp3 gene can be introduced into a subject^'s cells by lipofection in \ ιvo using hposomes. Synthetic catio c hpids can be used to prepare hposomes for m

o transfection of a gene encoding a marker (Feigner el al, Proc. Nat 'l Acad. Sa. USA 54:7413 ( 1987). Mackey et al. Proc. Nat 'l Acad. Sci USA 85:8027 ( 1988)). The use of lipofection to introduce exogenous genes into specific organs m

o has certain practical advantages. Liposomes can be used to direct transfection to particular cell types, which is particulailv advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other molecules for the purpose of targeting Targeted peptides (e.g.. hormones or neurotransmitteis). proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically

Electroporation is another alternative mode of administration of a Ztryp3 nucleic acid molecules For example, Aihara and Miyazaki. Nature Biotechnology 16:867 ( 1998), have demonstrated the use of vivo electroporation for gene transfer into muscle.

In an alternative approach to gene therapy , a therapeutic gene may encode a Ztryp3 anti-sense RNA that inhibits the expression of Ztryp3. Methods of preparing anti-sense constructs are known to those in the art See, for example. Erickson et al, Dew Genet. 74.274 (1993) [transgemc mice]. Augustine et al., Dew Genet. 14:500 (1993) [muπne whole embryo culture], and Olson and Gibo, Exp. Cell Res. 241: 134 (1998) [cultured cells]. Suitable sequences for Ztryp3 anti-sense molecules can be derived from the nucleotide sequences of Ztryp3 disclosed herein. Alternatively, an expression vector can be constructed in which a regulatory element is operably linked to a nucleotide sequence that encodes a πbozyme. Ribozymes can be designed to express endonuclease activity that is directed to a certain target sequence in a mRNA molecule (see, for example. Draper and Macejak, U.S. Patent No. 5,496.698, McSwiggen, U.S Patent No. 5.525,468. Chowπra and McSwiggen, U.S. Patent No. 5.631.359, and Robertson and Goldberg. U.S. Patent No. 5.225,337) In the context of the present invention, ribozymes include nucleotide sequences that bind with Ztryp3 mRNA.

In another approach, expression vectors can be constructed in which a regulatory element directs the production of RNA transcπpts capable of promoting RNase P-mediated cleavage of mRNA molecules that encode a Ztryp3 gene. According to this approach, an external guide sequence can be constructed for directing the endogenous πbozyme. RNase P. to a particular species of intracellular mRNA. which is subsequently cleaved by the cellular πbozyme (see, for example Altman et aϊ U S Patent No 5 168,053. Yuan et al Science 263 1269 ( 1994) Pace et al international publication No WO 96/18733, George et al , international publication No WO 96/21731. and W erner et al international publication No WO 97/33991 ) Preferably, the external guide sequence comprises a ten to fifteen nucleotide sequence complementarv to Ztrvp^ mRNA, and a 3'-NCCA nucleotide sequence, wherein N is preferably a puπne Tne external guide sequence transcπpts bind to the targeted mRNA species by the formation of base pairs between the mRNA and the complementary external guide sequences thus promoting cleavage of mRNA by RNase P at the nucleotide located at the 5 -side of the base-paired region

In general the dosage of a compos ition comprising a therapeutic v ectoi having a Ztryp3 nucleotide acid sequence such as a recombinant virus w ill v aiv depending upon such factors as the subject s age w eight, height, sex genei al medical condition and previous medical history Suitable routes of administration of therapeutic vectors include intravenous injection, intraarterial injection lntrapeπtoneal injection intramuscular injection, intratumoral injection, and injection into a cavity that contains a tumor

A composition comprising viral vectors non-viral vectors, or a combination of viral and non-viral vectors of the present inv ention can be formulated according to known methods to prepare pharmaceutically useful compositions whereby vectors or viruses are combined in a mixtuie with a pharmaceutically acceptable carrier As noted above, a composition, such as phosphate-buffered saline is said to be a ' pharmaceutically acceptable carrier" if its administration can be tolerated bv a recipient subject Other suitable carriers are w ell-known to those in the art (see. tor example, Remington s Pharmaceutical Sciences. 19th Ed (Mack Publishing Co 1995 ) and Gilrnan s the Pharmacological Basis of Therapeutics 7th Ed (MacMillan Publishing Co 1985))

For purposes of therapy, a therapeutic gene expression v ector, or a recombinant virus comprising such a vector, and a pharmaceutically acceptable carrier are administered to a subject in a therapeutically effective amount A combination of an expression vector (or virus) and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient subject W hen the subject treated with a therapeutic gene expression v ector or a recombinant virus is a human, then the therapy is preferably somatic cell gene therapv That is the preferred treatment of a human with a therapeutic gene expression v ector or a recombinant virus does not entail introducing into cells a nucleic acid molecule that can form part of a human germ line and be passed onto successi e generations ( i e human germ line gene therapy)

14. Production of Transgenic Mice

Transgemc mice can be engineered to over-express the Ztrxp3 gene in all tissues oi under the control of a tissue-specific or tissue-preferred regulatory element These ov er-producers of Ztryp3 can be used to characterize the phenotype that results from over-expression, and the transgenic animals can serv e as models for human disease caused by excess Ztryp3 Transgenic mice that ov er express Ztryp3 also piov ide model bioieactors for pioduction of Ztryp3 in the milk or blood of larger animals Methods for producing transgenic mice are w ell-know n to those ot skill in the ai t ( see for example. Jacob, "Expression and Knockout of Interferons in Transgenic Mice in Over expr ession and Knockout of Cy tokines in Tr ansgenic Mice. Jacob (ed ) pages 1 1 1 - 124 (Academic Press, Ltd 1994) Monastersky and Robl (eds ), Strategies m Transgenic Animal Science (ASM Press 1995), and Abbud and Nilson, "Recombinant Protein Expression in Transgenic Mice." in Gene Expression System s Using Nature for the Art of Expression, Fernandez and Hoeffler (eds ), pages 367-397 (Academic Press, Inc 1999)) For example, a method fo' pi oducing a transgenic mouse that expresses a Ztrxp3 gene can begin with adult fertile males (studs) (B6C3fl . 2-8 months of age (Taconic Farms, Germantown, NY)), vasectomized males (duds) (B6D211, 2-8 months, (Taco c Farms)), prepubescent fertile females (donois) (B6C3fl . 4-5 weeks (Taconic Farms)) and adult fertile females (recipients) (B6D2fl , 2-4 months. (Taconic Farms)) The donors are acclimated for one week and then injected with approximately 8 IU/mouse of Pregnant Mare^'s Serum gonadotrophin (Sigma Chemical Company, St Louis, MO) I P . and 46-47 hours later, 8 IU/mouse of human Choriomc Gonadotropm (hCG (Sigma)) I P to induce superovulation Donors are mated with studs subsequent to hormone injections Ovulation generally occurs within 13 houis of hCG injection Copulation is confirmed by the presence of a vaginal plug the morning following mating

Fertilized eggs aie collected under a surgical scope The ov iducts are collected and eggs are released into urmanalysis slides containing hyaluromdase (Sigma) Eggs aie washed once in hyaluromdase. and twice in Whitten^'s W640 medium (described, for example, by Memno and O'Claray . Biol Reprod 77 159 (1986), and Dienhart and Downs. Zy gote 4 129 ( 1996) ) that has been incubated with 57c CO... 57c O,, and 90%- l at 37°C The eggs are then stored in a 37 C/57c CO, incubator until microinjection

Ten to twenty micrograms of plasmid DN A containing a Ztryp3 encoding sequence is linearized, gel-purified, and resuspended in 10 mM Tπs-HCl (pH 7 4) 0 25 mM EDTA (pH 8 0) at a final concentration of 5- 10 nanograms per microhter for microinjection For example, the Ztryp3 encoding sequences can encode a polypeptide comprising amino acid residues 20 to 235 of SEQ ID NO 2

Plasmid DNA is microinjected into harvested eggs contained in a diop of W640 medium overlaid by warm CO.. -equilibrated mineral oil The DNA is drawn into an injection needle (pulled from a 0 75mm ID 1mm OD borosihcate glass capillary ) and injected into individual eggs Each egg is penetrated w ith the injection needle into one or both of the haploid pronuclei

Picohters of DN A are injected into the pronuclei and the injection needle withdraw n without coming into contact with the nucleoh The procedure is repeated until all the eggs are injected Successfully microinjected eggs are transferred into an organ tissue-culture dish with pre-gassed W640 medium for storage overnight in a 37°C/57c CO, incubator

The following day, two-cell embryos are transferred into pseudopregnant recipients The recipients are identified by the presence of copulation plugs, after copulating with vasectomized duds Recipients aie anesthetized and shaved on the dorsal left side and transferred to a surgical microscope A small incision is made in the skin and through the muscle wall in the middle of the abdominal aiea outlined by the πbcage. the saddle, and the hind leg, midway betw een knee and spleen The reproductive organs are exteriorized onto a small surgical drape The fat pad is stretched out over the surgical drape, and a baby serrefine (Roboz. Rockville. MD) is attached to the fat pad and left hanging over the back of the mouse, preventing the organs from sliding back in

With a fine transfer pipette containing mineral oil followed by alternating W640 and air bubbles, 12-17 healthy two-cell embryos from the previous day's injection are transferred into the recipient The swollen ampulla is located and holding the oviduct between the ampulla and the bursa. a nick in the oviduct is made with a 28 g needle close to the bursa, making sure not to tear the ampulla or the bursa

The pipette is transferred into the nick in the oviduct, and the embryos are blow n in, allowing the first air bubble to escape the pipette The fat pad is gently pushed into the peritoneum, and the reproductive organs allowed to slide in The peritoneal wall is closed with one suture and the skin closed with a wound clip The mice recuperate on a 37°C slide w armer for a minimum of four hours The recipients aie returned to cages in pairs and allowed 19 21 dav s gestation After birth 19 21 day s postpartum is allowed before weaning The weanlings aie sexed and placed into separate sex cages, and a 0 5 cm biopsv (used foi genotyping) is snipped off the tail with clean scissors Genomic DNA is prepared fiom the tail snips using, for example a

QIAGEN DNEASY kit follow ing the manufacture s instructions Genomic DNA is analyzed bv PCR using pπmeis designed to amplify a Ztr yp3 gene oi a selectable markei gene that was introduced in the same plasmid Aftei animals are confirmed to be transgenic, they aie back-crossed into an inbred strain bv placing a transgenic female w ith a w ild-tvpe male, or a transgenic male w ith one or tw o wild-type female(s) A pups aie born and weaned the sexes aie separated and their tails snipped foi genotvpmg

To check for expression of a tiansgene in a liv e animal a partial hepatectomv is performed A surgical prep is made of the upper abdomen directlv below the zyphoid process Using sterile technique a small 1 5-2 cm incision is made below the sternum and the left lateral lobe of the liver exteriorized Using 4 0 silk, a tie is made around the lower lobe securing it outside the body cav ity An atraumatic clamp is used to hold the tie while a second loop of absorbable Dexon (American Cyanamid Wayne, N J ) is placed proximal to the first tie A distal cut is made from the Dexon tie and approximately 100 mg of the excised liver tissue is placed in a sterile petπ dish The excised liver section is transferred to a 14 ml polypropylene round bottom tube and snap frozen in liquid nitrogen and then stored on dry ice The surgical site is closed with suture and wound clips, and the animal s cage placed on a 37°C heating pad for 24 hours post operatively The animal is checked daily post operatuely and the wound clips removed 7-10 days after surgery The expression level of Ztryp3 mRNA is examined for each transgenic mouse using an RNA solution hybridization assav or polymerase chain reaction

In addition to producing transgenic mice that over-express Ztryp3, it is useful to engineer transgenic mice with either abnormally low or no expression of the gene Such transgenic mice provide useful models for diseases associated with a lack of Ztryp3 As discussed abov e. Ztr yp3 gene expression can be inhibited using anti- sense genes πbozyme genes, or external guide sequence genes To produce transgenic mice that under-express the Zti yp3 gene such inhibitory sequences are targeted to Ztryp3 mRNA Methods for producing transgenic mice that hav e abnormally low expression of a particular gene are known to those in the art (see, for example, Wu et al "Gene Underexpression in Cultured Cells and Animals by Antisense DNA and RNA Strategies," in Methods in Gene Biotechnology , pages 205-224 (CRC Press 1997)).

An alternative approach to producing transgenic mice that have little or no Ztrxp3 gene expression is to generate mice having at least one normal Ztr\p3 allele replaced by a nonfunctional Ztryp3 gene One method of designing a nonfunctional Ztryp3 gene is to insert another gene, such as a selectable marker gene, within a nucleic acid molecule that encodes Ztryp3 Standard methods for producing these so-called "knockout mice" are known to those skilled in the art (see, for example, Jacob, "Expression and Knockout of Interferons in Transgenic Mice,^" in Overexpiesswn and Knockout erf Cytokmes in Transgenic Mice. Jacob (ed ), pages 1 1 1-124 (Academic Press. Ltd 1994), and Wu et al , "New Strategies foi Gene Knockout,^" in Methods Gene Biotechnology , page 339-365 (CRC Press 1997))

Claims

What is claimed is

1 An isolated polypeptide, comprising an amino acid sequence selected from the group consisting of amino acid residues 20 to 235 of SEQ ID NO 2. ammo acid residues 20 to 130 of SEQ ID NO 2. amino acid residues 20 to 198 of SEQ ID NO 2 and amino acid residues 40 to 198 of SEQ ID NO 2

2 The isolated polypeptide of claim 1 w herein the polypeptide compπses amino acid residues 20 to 235 of SEQ ID NO 2

3 The isolated polypeptide of claim 2 w herein the polvpeptide compnses amino acid iesidues 1 to 235 of SEQ ID NO 2

4 An isolated nucleic acid molecule wherein the nucleic acid molecule is a nucleic acid molecule that remains hybridized following stringent wash conditions to a nucleic acid molecule selected from the group consisting of (a) a nucleic acid molecule consisting of the nucleotide sequence of nucleotides 42 to 746 of SEQ ID NO 1 , (b) a nucleic acid molecule consisting of the nucleotide sequence of nucleotides 99 to 746 of SEQ ID NO 1. and (c) a nucleic acid consisting of a nucleotide sequence that is a complement of the nucleotide sequence of nucleic acid molecule (a) or (b)

5 An isolated nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 20 to 235 of SEQ ID NO 2, amino acid residues 20 to 130 of SEQ ID NO 2. amino acid residues 20 to 198 of SEQ ID NO 2. and amino acid residues 40 to 198 of SEQ ID NO 2

6 The isolated nucleic acid molecule of claim 5 wherein the nucleic acid molecule encodes a polypeptide comprising amino acid residues 20 to 235 of SEQ ID NO 2

7 The isolated nucleic acid molecule of claim 6 wherein the nucleic acid molecule comprises nucleotides 99 to 746 of SEQ ID NO 1

8 The isolated nucleic acid molecule of claim 5. wherein the nucleic acid molecule encodes a polypeptide compπsing amino acid residues 1 to 235 of SEQ ID NO 2

9 The isolated nucleic acid molecule of claim 8. wherein the nucleic acid molecule comprises nucleotides 42 to 746 of SEQ ID NO: 1 .

10 A vector, compπsing the isolated nucleic acid molecule of claim 5

1 1 A vector, comprising the isolated nucleic acid molecule of claim 6

12 An expression vectoi. comprising the isolated nucleic acid molecule of claim 6. a transcription promoter, and a transcription terminator, wherein the promoter is operably linked with the nucleic acid molecule, and w herein the nucleic acid molecule is operably linked w ith the transcription terminatoi

13 A recombinant host cell comprising the expression ectoi of claim 12. wherein the host cell is selected from the group consisting of bacterium, yeast cell, fungal cell, insect cell, mammalian cell, avian cell, and plant cell.

14 A method of using the expression vector of claim 12 to produce a polypeptide that comprises amino acid residues 20 to 235 of SEQ ID NO:2. comprising culturing recombinant host cells that comprise the expression vector and that produce the polypeptide.

15. The method of claim 14. further comprising isolating the polypeptide from the cultuied recombinant host cells

16. An antibody or antibody fragment that specifically binds with the polypeptide of claim 1

17. A composition, comprising a carrier and the polypeptide of claim 1.

18. A fusion protein, comprising the polypeptide of claim 1.

19. A method of detecting in a biological sample the presence of RNA that encodes the amino acid sequence of SEQ ID NO:2. comprising:

(a) contacting a nucleic acid probe under hybridizing conditions w ith either (i) test RNA molecules isolated from the biological sample, or (n) nucleic acid molecules synthesized from the isolated RNA molecules, wherein the probe has a nucleotide sequence comprising either a portion of the nucleotide sequence of nucleotides 42 to 746 of SEQ ID NO 1, or its complement, and

(b) detecting the formation of hybrids of the nucleic acid probe and either the test RNA molecules or the synthesized nucleic acid molecules wherein the presence of the hybrids indicates the presence of RNA that encodes the amino acid sequence of SEQ ID NO 2 in the biological sample

20 A method of detecting in a biological sample the presence of a polypeptide that comprises the amino acid sequence of amino acid residues 20 to 235 of SEQ ID NO 2, comprising

(a) contacting the biological sample with an antibody or an antibody fragment, of claim 16 wherein the contacting is performed under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and

(b) detecting any of the bound antibodv or bound antibody fragment