WO2005080560A1 - Mutant tace catalytic domain - Google Patents

Abstract

The invention relates to mutant TACE nucleic acids and amino acid sequences and methods for expressing TACE nucleic acids in host cells, such as prokaryotes.

Description

MUTANT TACE CATALYTIC DOMAIN

BACKGROUND OF THE INVENTION ADAM- 17 (TACE; tumor necrosis factor-alpha converting enzyme) can cleave cell bound TNFα, a type II membrane protein of relative molecular mass 26,000 (26 kD), to produce a soluble 17 kD form of TNF-α. The 17 kD form of TNF-α has been implicated in the inflammatory response seen in sepsis and septic shock (Spooner et al., Clinical Immunology and Immunopathology, 62: Sll (1992)) as well in rheumatoid arthritis. Several therapeutic proteins that bind to TNFα and ameliorate its pathological effects have been approved for the treatment of rheumatoid arthritis- ENBREL® (etanercept), REMICADE® (infliximab), and HUMIRA® (adalimumab). In addition, there is interest in developing small molecule inhibitors of TACE as human therapeutics (see e.g., Moss et al. (2001) Drug Discov. Today 6(8):417-426; Beck et al. (2002) J. Pharm. and Exper. Ther. 302: 390-396; and WO 00/09492). Thus, there is a need in the art for compounds and tools that can be used to identify TACE inhibitors for the treatment of diseases that involve TNFα.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides for an isolated nucleic acid comprising: a nucleotide sequence encoding a polypeptide of formula X-J-Z, wherein X is a prokaryotic secretion signal sequence; wherein J is a TACE catalytic domain lacking a TACE pro domain; wherein said TACE catalytic domain has from 68% to 100% amino acid identity to the entire length of SEQ ID NO: 2, Z is absent or a tag domain; and wherein said X-J-Z polypeptide can cleave a polypeptide comprising SEQ ID NO: 17 between the Ala and Nal residues. In certain embodiments, J comprises SEQ ID NO: 25. In other embodiments, J comprises one to seven SEQ ID NOS selected from the group consisting of: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32. In still other embodiments, the polypeptide of formula X-J-Z is SEQ ID NO: 6. In certain embodiments, J is SEQ ID NO: 4 or SEQ ID NO: 2. In still other embodiments, the prokaryotic secretion signal sequence is SEQ ID NO: 16. Z is a tag domain in certain embodiments. An example of Z is SEQ ID NO: 14. In certain embodiments, the nucleic acid is SEQ ID NO: 5. In another aspect, the invention relates to polypeptides comprising: a polypeptide of formula X-J-Z, wherein X is a prokaryotic secretion signal sequence; wherein J is a TACE catalytic domain lacking a TACE pro domain, wherein said TACE catalytic domain has from about 68% to about 100% amino acid identity over the entire length of SEQ ID NO: 2; Z is absent or a tag domain; and wherein said X-J-Z polypeptide can cleave a polypeptide comprising SEQ ID NO: 17 between the Ala and Val residues. In certain embodiments, the polypeptide comprises SEQ ID NO: 4 or SEQ ID NO: 2. In still other embodiments, X is SEQ ID NO: 16. Z is a tag domain in certain embodiments. An example of Z is SEQ ID NO: 14. In certain embodiments, the polypeptide of formula X-J-Z comprises SEQ ID NO: 6. In another aspect, the present invention provides for expression vectors comprising: a prokaryotic promoter operably linked to a nucleic acid comprising: a nucleotide sequence encoding a polypeptide of formula X-J-Z, wherein X is a prokaryotic secretion signal sequence; wherein J is a TACE catalytic domain lacking a TACE pro domain, wherein said TACE catalytic domain has from 68% to 100% amino acid identity over the entire length of SEQ ID NO: 2, Z is absent or a tag domain; and wherein said X-J-Z polypeptide can cleave a TACE substrate. In certain embodiments, the polypeptide of formula X-J-Z comprises SEQ ID NO: 4 or SEQ ID NO: 2. In still other embodiments, X is SEQ ID NO: 16. Z is a tag domain in certain embodiments. An example of Z is SEQ ID NO: 14. In certain embodiments, the polypeptide of formula X-J-Z comprises SEQ ID NO: 6. In still another aspect, the present invention provides for a prokaryotic cell comprising: an expression vector comprising a promoter operably linked to a nucleic acid comprising: a nucleotide sequence encoding a polypeptide of formula X-J-Z, wherein X is a prokaryotic secretion signal sequence; wherein J is a TACE catalytic domain lacking a TACE pro domain, wherein said TACE catalytic domain has from 68% to 100% amino acid identity over the entire length of SEQ ID NO: 2, Z is absent or a tag domain; and wherein said X-J-Z polypeptide can cleave a TACE substrate. In certain embodiments, J is SEQ ID NO: 4 or SEQ ID NO: 2. In other embodiments, the nucleotide sequence encoding a polypeptide of formula X-J-Z is SEQ ID NO: 6. In certain embodiments, the polypeptide of formula X-J-Z comprises SEQ ID NO: 4 or SEQ ID NO: 2. In still other embodiments, X is SEQ ID NO: 16. Z is a tag domain in certain embodiments. An example of Z is SEQ ID NO: 14. In certain embodiments, the polypeptide of formula X-J-Z comprises SEQ ID NO: 6. In certain embodiments, the cell is an E. coli cell. In still another aspect, the present invention provides for methods comprising: growing a culture of prokaryotic cells, wherein said cells comprise: an expression vector comprising a promoter operably linked to a nucleic acid comprising: a nucleotide sequence encoding a polypeptide of formula X-J-Z, wherein X is a prokaryotic secretion signal sequence; wherein J is a TACE catalytic domain lacking a TACE pro domain, wherein said TACE catalytic domain has from 68% to 100% amino acid identity over the entire length of 5 SEQ ID NO: 2, Z is absent or a tag domain; and wherein said X-J-Z polypeptide can cleave a TACE substrate; and purifying said X-J-Z polypeptide or a X-J-Z polypeptide where X has been proteolytically removed. In certain embodiments, J is SEQ JD NO: 4 or SEQ ID NO: 2. In certain embodiments, the polypeptide of formula X-J-Z comprises SEQ ID NO: 4 or SEQ ID NO: 2. Z is a tag domain in certain embodiments. An example of Z is SEQ ID NO: 14. 10 In certain embodiments, the polypeptide of formula X-J-Z comprises SEQ ID NO: 6. In certain embodiments, the culture is grown at 16-20 °C. In certain embodiments, the X-J-Z polypeptide or X-J-Z polypeptide where X has been proteolytically removed is purified without the addition of a chaotropic agent or a cystine disulfide bond reducing agent. In certain embodiments, the prokaryotic cells are E. coli cells.

15 BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts a nucleotide sequence of a nucleic acid encoding a mutant TACE catalytic domain (SEQ ID NO: 3) (top line) and the amino acid sequence encoded by SEQ ID NO: 3 (SEQ ID NO: 4) (lower line). Figure 2 depicts an alignment of polypeptide sequences for a mutant TACE catalytic

20 domain (top polypeptide sequence; SEQ ID NO: 4), and a human TACE catalytic domain (bottom polypeptide sequence; SEQ ID NO: 2). The sequences were aligned using a blastp program for two polypeptides on the National Center for Biotechnology Information (NCBI) website (Tatusova et al., (1999) FEMS Microbiol. Lett. 174:247-250) with a Matrix of BLOSUM62, an Open gap of 11, an extension gap of 1, a gap x_dropoff of 50, an expect of

25 10.0, a word size of 3, and the filter on.

DEFINITIONS

A "TACE catalytic domain" is a polypeptide that has the ability to cleave a TACE substrate comprising the sequence LAQAVRSSSR (SEQ ID NO: 17) between the Ala and Val residues. In certain embodiments, a TACE catalytic domain is no longer than SEQ ID 30 NO: 2. An example of a TACE catalytic domain is SEQ ID NO: 2. A "TACE pro domain" is a domain of a native TACE polypeptide between the TACE signal sequence and the beginning of the TACE catalytic domain. An example of a TACE pro domain is SEQ ID NO: 19. In native mammalian cells, the native pro domain is typically cleaved off during its processing to the mature form of TACE. An example of a TACE signal sequence is SEQ ID NO: 20. A "TACE catalytic domain lacking a TACE pro domain" is a TACE catalytic domain that is not contiguous with a TACE pro domain. Examples of "TACE catalytic domains lacking a TACE pro domain" include SEQ ID NO: 19 and SEQ ID NO: 4. A "tag domain" is a polypeptide sequence that when fused to another protein facilitates the purification of the resulting fusion protein. Examples of tag domains include GST (glutathione S-transferase) and a poly-histidine tag (e.g., SEQ ID NO: 14). A "candidate therapeutic agent is able to inhibit TACE activity" if the agent can decrease a TACE polypeptide' s proteolytic activity. An "expression vector" is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter. The term "transfect" or "transduce" refers to any manner of introducing a nucleic acid into a cell including electroporation, plasmid transfection, viral transduction, lipid-nucleic acid complexes, naked DNA, etc. "Inhibitors" of TACE or "TACE inhibitory compounds" are compounds that decrease TACE activity or another TACE activity, e.g., antagonists, ligands, etc. Samples or assays comprising TACE that are treated with a potential inhibitor are compared to control samples without the inhibitor. The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or by the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell or over-express genes that are found within the native form of the cell. A "promoter" is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. A promoter also optionally includes distal enhancer or repressor elements. A "prokaryotic promoter" is a promoter that is active in a prokaryote (e.g., E. coli) such that it when operably linked to a second nucleic acid, can direct the transcription of the second nucleic acid. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence. A TACE polypeptide of the present invention is "purified" or "isolated" if it is at least 51% (w/v). Examples of purified TACE polypeptides of the present invention include preparations that are at least 51% (w/v), preferably 85% (w/v), more preferably at least 95% (w/v), and most preferably at least 99% (w/v) of the preparation when purified from a prokaryotic or eukaryotic cell population. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A TACE nucleic acid is isolated if it is separated from the open reading frames that flank a native endogenous TACE gene. "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, peptide-nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term "nucleic acid" is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences. The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., on a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group (e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium). Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known 3 -letter symbols or by the 1 -letter symbols recommended by the IUPAC-IUB Biochemical

Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The term "identity" in the context of 2 nucleic acids or polypeptides, refers to the number of identical residues that a first contiguous sequence has when compared and aligned for maximum correspondence to a second contiguous sequence, as measured using a sequence comparison software program or by visual inspection. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a computerized sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated if changes from the default parameters are desired. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math., 1981;2:482, by the homology alignment algorithm of Needleman & Wunsch, /. Mol. Biol., 1970;48:443, by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA., 1988;85:2444), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), GeneDoc Program, Version 2.6.001, or by visual inspection (see generally, Current Protocols in Molecular Biology, Ausubel et al., eds., 1998). Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol, 1990;215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

DETAILED DESCRIPTION

I. INTRODUCTION The present invention relates to TACE nucleic acids and proteins, methods for expressing TACE proteins, and methods for the use of TACE proteins and nucleic acids, etc. As used herein, the term "TACE polypeptide" or "TACE protein" includes a protein or polypeptide that encodes a metalloprotease capable of cleaving the Ala-Val bond in a polypeptide comprising SEQ ID NO: 17.

II. ISOLATION OF TACE ENCODING NUCLEIC ACIDS Nucleic acids encoding TACE polypeptides of interest (e.g., TACE catalytic domains) can be made or isolated using routine techniques in the field of recombinant genetics and synthetic nucleic acid chemistry. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., 1989; Kriegler, Gene Transfer and Expression: A Laboratory Manual, 1990; and Current Protocols in Molecular Biology, Ausubel et al., eds., 1998. In general, TACE catalytic domain sequences can be cloned from cDNA and genomic

DNA libraries by hybridization with a probe, or isolated using nucleic acid amplification techniques with oligonucleotide primers using methods that are known in the art. For example, TACE catalytic domain sequences can be isolated from mammalian nucleic acid (e.g., cDNA) libraries by hybridizing with a nucleic acid probe, the sequence of which can be derived from SEQ ID NO: 1 or other TACE nucleic acids (see e.g., U.S. Patent No. 5,830,742; Black et al (1997) Nature 385: 729-33; Moss et al (1997) Nature 385: 733-6; Rosendahl et al. (1997) J. Biol. Chan. 272: 24588-24593; Clarke et al (1998) Prot. Exp. & Purif. 13: 104-11; GenBank Accession Nos. U69611 (SEQ ID NO: 33); and U69614 (SEQ ID NO: 34)). Nucleic acid amplification techniques (e.g., the polymerase chain reaction ("PCR") using primers can also be used to amplify and isolate, e.g., a nucleic acid encoding TACE, from DNA or RNA (see, e.g., Dieffenbach & Dveksler, PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory, 1995). These primers can be used, e.g., to amplify either a TACE nucleic acid sequence or a probe of one to several hundred nucleotides (e.g., SEQ ID NO: 1). Nucleic acids amplified by the PCR can be subcloned into an appropriate vector. Synthetic oligonucleotides can also be used to construct recombinant genes for use as probes or for expression of protein. This method is performed using a series of overlapping oligonucleotides usually 40 to 120 bp in length, representing both the sense and nonsense strands of the gene. These DNA fragments are then annealed, ligated, and cloned. The specific subsequence may then be ligated into an appropriate vector (e.g., a shuttle vector or an expression vector). Alternatively, the entire TACE catalytic domain nucleic acid can be synthesized as one contiguous nucleic acid. Because of the degeneracy of the genetic code, a large number of functionally equivalent nucleic acids can encode a given TACE polypeptide sequence. For instance, the codons GCA, GCC, GCG, and GCT all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. In addition, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 1991;19:5081; Ohtsuka et al., J. Biol. Chem., 1985;260:2605-2608; Rossolini et al., Mol. Cell. Probes, 1994; 8: 91-98). These and other mutant TACE nucleotide sequences can be generated using methods known in the art, such as site-directed mutation technology, PCR, LCR, ligation of overlapping oligonucleotides, etc. Other mutant TACE nucleic acids (e.g., SEQ ID NO: 3) can be generated that encode polypeptides with about 68% amino acid identity to SEQ ID NO: 2. These TACE nucleic acids can encode a TACE catalytic domain polypeptide that lacks a TACE pro domain and that comprises SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ DD NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and or SEQ ID NO: 32.

In certain embodiments, a fusion protein comprising a tag domain (e.g., a poly- histidine tag) fused directly or through a linker region to a TACE polypeptide can be expressed in a prokaryote or eukaryote. Polynucleotide vectors that facilitate the expression of fusion proteins are commercially available (e.g., New England Biolabs, Invitrogen and Novagen). For example, a polyhistidine tag (e.g., SEQ ID NO: 14 - GSHHHHHH) can be fused to a TACE amino acid sequence by subcloning a TACE sequence into a pET24 or pET28 vector (Novagen, Madison, WI). A polyhistidine tagged-TACE polypeptide can be expressed in E. coli and purified over a nickel affinity column by eluting with imidazole or another suitable eluant. Alternatively, a nucleic acid encoding a glutathione S-transferase (GST) domain can be fused to a nucleic acid encoding a TACE catalytic domain of the present invention and then expressed in a prokaryotic organism, such as E. coli (see generally (Smith and Johnson (1988) Gene 67: 31-40; Guan and Dixon (1991) Anal. Biochem. 192: 262-267). The GST TACE catalytic domain fusion protein can then be purified over a glutathione agarose column. In certain embodiments, a GST domain is fused to the N- terminal end of a polypeptide that codes for a TACE of the present invention.

III. EXPRESSION OF TACE NUCLEIC ACIDS The nucleic acid encoding the TACE polypeptide of choice can be cloned into expression vector before transformation into prokaryotic or eukaryotic cells for replication and/or expression. Suitable prokaryotic expression vectors are known in the art and described, e.g., in Sambrook et al., supra., 1989 and Ausubel et al., supra., 1998, and include the pET vectors. Bacterial expression systems for expressing TACE proteins are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene, 1983;22:229-235). Kits for such expression systems are also commercially available. Recombinant TACE proteins can be purified for use in enzyme assays, and for use in generating TACE antibodies. Recombinant TACE can be purified from any suitable expression system, e.g., by expressing a TACE protein in a host and then purifying the recombinant protein via affinity purification. In some embodiments, the recombinant protein is a fusion protein that contains a tag that facilitates purification, e.g., a polyhistidine tag. TACE proteins can be also separated from other proteins and components of the source material, (e.g., a recombinant cell) by standard separation techniques, e.g., column chromatography. Protein purification techniques include, but are not limited to, solubility fractionation (e.g., precipitation with ammonium sulfate, etc.), immunoprecipitation, centrifugation, ultracentrifugation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, and immunoaffinity chromatography, (see e.g., Scopes, Protein Purification: Principles and Practice, Springer- Verlag New York Inc., 1982). Typically one or more of these techniques can be used to enrich for or isolate a TACE polypeptide. In certain embodiments, a nucleic acid encoding a prokaryotic secretion signal sequence is linked to a nucleic acid encoding a TACE catalytic domain. The resulting nucleic acid encodes a prokaryotic secretion signal polypeptide sequence linked to a TACE catalytic domain polypeptide sequence. A "prokaryotic secretion signal sequence" is an amino acid sequence that when fused to a second polypeptide can direct the expression of TACE polypeptide it is fused to into the periplasmic space of a prokaryote and in some cases outside the cell. Typically, prokaryotic secretion signal sequences are from 10-40 amino acids in length. In certain embodiments, prokaryotic secretion signal sequences are from 20-30 amino acids in length. Signal sequences are predominantly comprised of hydrophobic amino acids. In certain cases, the secretion signal sequence is proteolytically removed by the prokaryote during the secretion process. Examples of prokaryotic signal sequences are known in the art and include but are not limited to: an Escherichia coli α-amylase signal sequence (e.g., SEQ ID NO: 21), an Escherichia coli ompA signal sequence (e.g., SEQ ID NO: 22), an Escherichia coli alkaline phosphatase sequence (e.g., SEQ ID NO: 23), and a Bacillus subtilis α-amylase signal sequence (e.g., SEQ ID NO: 24) (see e.g., Watson et al. (1984) Nucleic Acids Research 12(13):5145-5164); Klein et al. (1992) Protein Eng. 5: 511-517; and Oka et al. (1985) Proc. Natl. Acad. Sci. USA 82: 7212-7216). An example of a prokaryotic secretion signal sequence is a pectate lyase (pelB) secretion signal sequence (e.g., SEQ ID NO: 16). The pelB signal sequences can be derived from the pectate lysase gene of the phytopathogenic bacteria Erwinϊa chrysanthemi and Erwinia carotovora (see e.g., Lei et al. (1987) J. Bacteriol. 169: 4379-4383; Keen et al. (1986) J. Bacteriol. 168(2):595-606; U.S. Patent Nos: 5,846,818 and 5,576,195). Typically, the prokaryotic cells containing a prokaryotic expression vector that encodes a polypeptide comprising a TACE catalytic domain will be cultured in an appropriate liquid medium. In certain embodiments, the culture is maintained at 16-20 °C for 1-24 hours of the time the culture is being grown. The expressed TACE catalytic domain polypeptide can then be purified from the culture and used, for example, in a TACE assay. In certain embodiments, where the TACE catalytic domain is fused to a prokaryotic secretion signal sequence, purification of active enzyme does not typically require the addition of a thiol- modifying reagent (e.g., 4-aminophenylmercuric acetate), a chaotropic agent (e.g., guanidine hydrochloride, urea, etc.) and/or a cystine disulfide bond reducing agent (e.g., glutathione, β- mercaptoethanol, dithiothreitol, etc.). In certain embodiments, an X-J-Z polypeptide and/or a X-J-Z polypeptide where X has been proteolytically removed is purified. In certain embodiments, the prokaryotic secretion signal sequence can be cleaved from the X-J-Z polypeptide by an endogenous protease or by an exogenously added protease to provide a polypeptide that comprises J and Z, but not X. Alternatively, a polypeptide comprising a TACE catalytic domain (e.g., SEQ JD NO: 4) may be expressed in eukaryotic cells. A variety of methods are also known in the art for expressing a gene in eukaryotes (Ausubel et al., supra., 1998). Cells can be transfected with an expression vector containing a TACE nucleic acid of interest (e.g., SEQ ID NO: 3) and a promoter. The promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter is preferably positioned about the same distance from the heterologous transcription start site, as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function. The promoter typically can also include elements that are responsive to transactivation, e.g., hypoxia responsive elements, Gal4 responsive elements, lac repressor responsive elements, and the like. The promoter can be constitutive or inducible, heterologous or homologous. In addition to the promoter, the expression vector typically contains a transcription unit or expression vector that contains all the additional elements required for the expression of the nucleic acid in host cells. A typical eukaryotic expression vector thus contains a promoter operably linked, e.g., to the nucleic acid sequence encoding a TACE polypeptide, and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. In addition to a promoter sequence, the expression vector can also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes. 'Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, ρAV009/A+, pMT010/A+ pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the S V40 early promoter, S V40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells. Some expression systems have markers that provide gene amplification such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a TACE polypeptide encoding nucleic acid sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters. Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra., 1989).

IV. METHODS FOR IDENTIFYING TACE INHIBITORY COMPOUNDS Methods for identifying TACE inhibitory compounds are described below. Compounds that inhibit TACE may be useful as pharmaceutical agents in the treatment of TACE -mediated disorders and conditions. TACE assays are carried out in the presence or absence of a TACE inhibitory compound and the amount of enzyme activity is compared for a determination of inhibitory activity of the TACE inhibitory compound. Samples that are not contacted with a TACE inhibitory compound are assigned a relative TACE activity value of 100. Inhibition of TACE activity is achieved when the TACE activity in the presence of a TACE inhibitory compound is less than the control sample (i.e., no inhibitory compound). Preferably, the TACE activity value relative to the control is less than 90%, more preferably less than 50%, and still more preferably less than 20%. The IC50 of a compound is the concentration of compound that inhibits 50% of the control sample activity. Typically, candidate therapeutic agents are screened to identify TACE inhibitory compounds, which can be further, tested for there ability to treat a TACE mediated disorder or condition. A. Candidate therapeutic agents. A "candidate therapeutic agent" is a compound that is being tested for its ability to inhibit a TACE polypeptide. Essentially any chemical compound can be screened as a TACE candidate therapeutic agent in a TACE assay to identify a TACE inhibitory compound. The compounds tested as inhibitors of TACE can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid. Typically, test compounds will be small chemical molecules and peptides. The candidate therapeutic agent can be a naturally occurring compound, one that is artificially synthesized, or one that is made by a combination these methods. In certain embodiments, the candidate therapeutic agents are organic molecules under 1000 Da, more preferably under 500 Da, and still more preferably are between 500 and 100 Da, and pharmaceutically acceptable salts thereof. Examples of TACE inhibitors can be found in WO 00/09492. In some embodiments, assays can be used to screen a combinatorial compound library that contains a large number of potential therapeutic compounds (potential inhibitor compounds). In some embodiments, compounds dissolved in aqueous or organic (e.g., DMSO-based) solutions are screened. "Combinatorial compound libraries" can be screened in one or more TACE assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics. A combinatorial compound library is a collection of diverse compounds that have been generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial compound library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). A plethora of compounds can be synthesized through such combinatorial mixing of chemical building blocks. The preparation and screening of combinatorial chemical libraries and other libraries of large numbers of compounds (e.g., over 1000 compounds) are well-known to those of skill in the art.

B. TACE Assays 5 Methods for assaying TACE activity have been described in the art. Typically, a sample containing a TACE polypeptide is combined with a protein or a synthetic peptide substrate and the proteolytic activity of TACE is monitored by measuring substrate depletion and/or product(s) formation. The use of synthetic peptide substrates for the determination of TACE catalytic activity are known in the art and are in some cases commercially available.

10 Examples of synthetic peptide TACE substrates include: (Abz-LAQAVRSSSR-Dap(Dnp)- NH₂ (Abz-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Dap(Dnp)-NH (SEQ ID NO: 35); 2- aminobenzoyl-L-Leucyl-L-Alanyl-L-Glutaminyl-L-Alanyl-L-Nalyl-L-Arginyl-L-Seryl-L- Seryl-L-Seryl-L-Arginyl-[Ν^ε-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl] amide; SDP- 3818-PI Peptides International, Inc., Louisville, KY, Jin et al. (2002) Anal. Biochem., 302:

15 269-275 (2002)); Dnp-SPLAQAVRSSSR-NH? (SEQ ID NO: 36) (Prod. No. M-2290, Bachem Corp.; Rosendahl et al (1997) JBiol Chem 272: 24588-24593); Mca-PLAQAN- Dap(Dnp)RSSSR-ΝH₂ (SEQ ID NO: 37) (Prod. No. M-2255, Bachem Corp., Dap = 2,3- diaminopropionic acid; Dnp = 2,4-dinitrophenyl, Nan Dyk et al (1997) Bioorg & Med Chem Lett 7(10): 1219-24; and Patel et al (1998) J Immunol 160: 4570-4579); acetyl-GE(EDAΝS)-

20 LAQAVRSSK(DABCYL)G-NH₂ (SEQ ID NO: 38) (Holms et al (2001) Bioorg & Med Chem Lett 11: 2907-2910); biotin-(Ahx)-SPLAQAVRSSSRT[³H]PS-NH₂ (SEQ ID NO: 39) (Ahx = aminohexanoic acid, Leesnitzer et al (2000) in High Throughput Screening for Novel Anti- Inflammatories; pages 87-100); DABCYL-LAOAVRSSSR-EDANS (SEQ ID NO: 40) (Prod. No. M-2155, Bachem Corp., Gearing et al (1994) Nature 370: 555-557, Mohler et al (1994)

25 Nature 370: 218-220); and LY-LAQAVRSSK(CMTR)R-CO₂H (SEQ ID NO: 41) (LY = Lucifer yellow, CMTR = 5-carboxytetramethylrhodamine, Geoghegan (1996) Bioconj. Chem 7: 385-391). TACE cleaves these substrates between the Ala and Val residues (see underlined AN in the above listed TACE substrates). In certain embodiments, a peptide substrate will comprise at least one detectable label

30 - such as a fluorescent label, a radioactive label, etc., which can be detected by appropriate means - e.g., fluorirneter, scintillation counter, etc. For example the peptide substrate Abz- LAQAVRSSSR-Dap(Dnp)-ΝH₂ (SEQ ID NO: 35) comprises a fluorophore Abz and a quencher Dpa (Jin et al., supra). TACE catalytic activity can be assayed for example on Abz- LAQAVRSSSR-Dap(Dnp)-NH₂ (SEQ ID NO: 35)by excitation at 320 nm and measuring the increase in fluorescence at 420 nm. Typically around 1-100 μM of substrate is used with varying amounts of a polypeptide with TACE activity (e.g., 1 nM -100 nM of a TACE polypeptide). In certain embodiments, the assay mixture can be subjected to chromatography, e.g., on an HPLC, that will separate the cleaved TACE substrate portions. The effluent can be monitored for a particular wavelength to measure the amount of processed substrate. In other embodiments, radioactive substrates can be employed (e.g., Biotin-(Ahx)- SPLAOAVRSSSRTr³HIPS-NH₂ (SEQ ID NO: 39)).

EXAMPLES It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

EXAMPLE 1 A nucleic acid sequence comprising SEQ ID NO: 5 (which encodes SEQ ID NO: 6), a

5' Nde I restriction site, and a 3' Xho I restriction site encoding was generated by Blue Heron Biotechnology, Inc. (Bothell, WA), using their GeneMaker™ technology. The construct was sub-cloned from the pUCl 19 vectors provided by Blue Heron into the Nde I and Xho I restriction sites of pET24 (Novagen) to provide pET24-TACEmut. The pET24-TACEmut was expressed in the E. coli BL21-AI (arabinose-inducible) cell line (Invitrogen, Product No. C6070-03) that was grown at 16 °C or 20 °C in 5 mL of Superbroth medium (K.D. Medical, Inc., Product No. BLF-7160), induced with 100-250 μM IPTG (isopropylthio-y&D-galactoside, Gibco BRL Product No. 15529-019) plus 0.2% L-(+)- arabinose (EM Science 1.01492.0100), and allowed to express the induced protein overnight. The cells were spun down into a pellet, resuspended in 5 ml of fresh Superbroth medium, and then lysed with the addition of 12.5 ml of PopCulture® reagent (Novagen, Product No. 71092-5). The PopCulture reagent used was supplemented with: 40U of lysozyme/mL of original culture, 25U of Benzonase Nuclease/mL of original culture, and A of an EDTA-free Roche Complete Cocktail Protease inhibitor tablet (Product No. l-873-580)/12.5 mL of PopCulture. The components were mixed and added as follows: lysis: 0.1 nιL/1 mL of culture of above freshly made PopCulture® (supplemented as described) was added, mixed and incubated for 15 min at room temperature. The resultant cell lysate (~5.5 mL) was passed over a Ni-charged NTA affinity resin (SwellGel Ni-NTA discs from Pierce Biotechnology, Inc., Rockford, IL, Product No. 27-2500-01). The SwellGel was washed three times with 2 mL each of 0.05 M sodium phosphate buffer (pH 7.5) containing 0.005 M imidazole and 0.3 M sodium chloride. The SEQ ID NO: 6 proteins were eluted with 0.25 M (in place of 0.005 M) imidazole in the above wash buffer. Samples of the 0.25 M imidazole eluates were subjected to SDS-PAGE analysis and immunoblotting. The proteins in the samples were separated by electrophoresis in duplicate on Bio-Rad Criterion Precast 4-20% polyacrylamide gels Tris-HCl buffer (Product No. 345- 00334) with 1.0 mm gel thickness, 26 lanes, and 15 μL sample volumes. After electrophoresis, one gel was stained with Coomassie Blue and the other gel was electro- transferred to nitrocellulose. Pierce Super Signal West Pico (Product No. 34079) and India His-Probe HRP-Pierce (Product No. 15165) were used according to the manufacturer's instructions to detect SEQ ID NO: 6 on the nitrocellulose blot. The calculated relative molecular masses (M_τ) for SEQ ID NO: 6 is 33,110 Da. Samples containing purified SEQ ID NO: 6 were assayed for TACE activity using a commercial synthetic fluorigenic peptide substrate, Abz-LAQAVRSSSR-Dpa-(Dnp)- NH₂ (Product No. SDP-3818-PI; Peptides International, Inc.; Louisville, KY), and for inhibition by a TACE inhibitor, 4-acetyl-3-methyl-l-[4-(2-methyl-benzyIoxy)- benzenesulfonyl]-piperazine-2-carboxylic acid hydroxyamide (see WO 00/09492). The assay mixture contained 50 mM Tris-HCl buffer (pH 7.5), 20 μM ZnCl₂, 10 μM substrate, and 39 μL of Ni-column purified SEQ ID NO: 6 enzyme (0.3 to 2.6 μg total protein used), 1% DMSO, and 0 or 10 μM of a TACE inhibitor - 4-acetyl-3-methyl-l-[4- (2-methyl-benzyloxy)-benzenesulfonyl]-piperazine-2-carboxylic acid hydroxyamide per 50-μL assay. The reaction was incubated at 37 °C and product formation was monitored continuously at 405 nm using a Molecular Devices _max 96- well plate fluorimeter (320 nm excitation filter) (Molecular Devices Corporation, Sunnyvale CA). TACE activity is expressed as relative fluorescent units (RFUVminute in Table 1. The initial rate, RFU/min, is defined as the slope of the initial linear part of the plot of fluorescence versus time. The specific activity of the Ni-NTA-purified SEQ ID NO: 6 samples is given in RFU/min/μg. Table 1

All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

CLAIMSWhat is claimed is:

1. An isolated nucleic acid comprising: a nucleotide sequence encoding a polypeptide of formula X-J-Z, wherein X is a prokaryotic secretion signal sequence; wherein J is a TACE catalytic domain lacking a TACE pro domain; wherein said TACE catalytic domain has from 68% to 100% amino acid identity to the entire length of SEQ ID NO: 2, Z is absent or a tag domain; and wherein said X-J-Z polypeptide can cleave a polypeptide comprising SEQ ID NO: 17 between the Ala and Val residues.

2. The nucleic acid of Claim 1, wherein J comprises SEQ ID NO: 25.

3. The nucleic acid of Claim 2, wherein J comprises one to seven SEQ ID NOS selected from the group consisting of: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ TD NO: 31, and SEQ ID NO: 32.

4. The nucleic acid of Claim 1, wherein J is SEQ ID NO: 4 or SEQ ID NO: 2.

5. The nucleic acid of Claim 2, wherein said prokaryotic secretion signal sequence is SEQ ID NO: 16, and wherein Z is SEQ ID NO: 14.

6. The nucleic acid of Claim 5, wherein said polypeptide of formula X-J-Z is SEQ ID NO: 6.

7. The nucleic acid of Claim 5, wherein said nucleic acid is SEQ ID NO: 5.

8. A polypeptide comprising: a polypeptide of formula X-J-Z, wherein X is a prokaryotic secretion signal sequence; wherein J is a TACE catalytic domain lacking a TACE pro domain, wherein said TACE catalytic domain has from about 68% to about 100% amino acid identity over the entire length of SEQ ID NO: 2; Z is absent or a tag domain; and wherein said X-J-Z polypeptide can cleave a TACE substrate.

5 9. The polypeptide of Claim 8, wherein J is SEQ ID NO: 4 or SEQ ID NO: 2.

10. The polypeptide of Claim 9, wherein X is SEQ ID NO: 16, and wherein Z is SEQ ID NO: 14.

11. The polypeptide of Claim 8, wherein said polypeptide of formula X-J-Z, is SEQ JD NO: 6.

10 12. A method comprising: growing a culture of prokaryotic cells, wherein said cells comprise: an expression vector comprising a promoter operably linked to a nucleic acid comprising: 15 a nucleotide sequence encoding a polypeptide of formula X-J-Z, wherein X is a prokaryotic secretion signal sequence; wherein J is a TACE catalytic domain lacking a TACE pro domain, wherein said TACE catalytic domain has from 68% to 100% amino acid identity over the entire length of SEQ ID NO: 2, 20 Z is absent or a tag domain; and wherein said X-J-Z polypeptide can cleave a polypeptide comprising SEQ ID NO: 17 between the Ala and Val residues; and purifying said X-J-Z polypeptide or a X-J-Z polypeptide where X has been proteolytically removed.

25 13. The method of Claim 12, wherein J is SEQ ID NO: 4 or SEQ ID NO: 2.

14. The method of Claim 13, wherein said nucleotide sequence encoding a polypeptide of formula X-J-Z, is SEQ JD NO: 6.

15. The method of Claim 12, wherein the culture is grown at 16-20 °C.

6. The method of Claim 12, wherein said X-J-Z polypeptide or X-J-Z polypeptide where X has been proteolytically removed is purified without the addition of a chaotropic agent or a cystine disulfide bond reducing agent.