JP2000501623A - Aspartic protease - Google Patents

Abstract

  (57) [Summary] The present invention relates to newly identified polynucleotides, polypeptides encoded by the polynucleotides, uses of the polynucleotides and polypeptides, and production of the polynucleotides and polypeptides. More specifically, the polypeptides of the present invention are believed to be novel aspartic proteases. The invention also relates to inhibiting the action of such polypeptides.

Description

DETAILED DESCRIPTION OF THE INVENTION Aspartic protease The present invention relates to newly identified polynucleotides, polypeptides encoded by the polynucleotides, uses of the polynucleotides and polypeptides, and production of the polynucleotides and polypeptides. More specifically, it is envisioned that the polypeptides of the present invention are novel aspartic proteases. The invention also relates to inhibiting the action of the polypeptide. Currently, five human aspartic proteases are known, namely, pepsin, gastricin, cathepsin D, cathepsin E, and renin, which have various functions. Pepsin and gastricin are involved in nutrient processes in the stomach, cathepsin D is involved in protein turnover in almost all cell types, and renin is highly involved in the production of angiotensin from the precursor angiotensinogen. It has a specific function. The exact role of cathepsin E has not been confirmed so far, but its presence in several epithelial cell types suggests a role in antigen processing. Cathepsin E may also be involved in certain inflammatory conditions, for example, Helicobacter pylori infection of the stomach. New aspartic proteases may have extracellular functions. There are a number of processes that may require an aspartic protease but for which the exact enzyme involved has not yet been identified. An important function implicated is the processing of endothelin and pro-opiomelanocortin prohormone. It is thought that aspartic protease is also involved in the processing of serum amyloid A protein. Aspartic proteases are so named because of the aspartic acid (Asp) residue pair required for hydrolytic cleavage of the peptide bond. The catalytic Asp residue is usually located within the -Hyd-Hyd-Asp-Thr-Gly-active site motif sequence, where Hyd is any hydrophobic amino acid. Eukaryotic aspartic proteases usually carry two such sequences, and these sequences have been shown by crystallography to be located adjacent to each other at the active site of the enzyme. these. Another highly conserved portion of the enzyme contains a β-hairpin loop structure (also called “flap”), which is above the active site and may aid in substrate binding. A novel aspartic protease was presented by Tang et al. Of the Okalahoma Medical Research Foundation at a presentation at the VIIth International Aspartic Protease Conference on October 22-27, 1996. According to one aspect of the present invention, there is provided a novel polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 15, or a fragment, analog or derivative thereof. The polypeptide of the present invention is of human origin. This polypeptide is believed to be an aspartic protease. The polypeptide of the present invention further comprises a polypeptide having the amino acid sequence of SEQ ID NO: 1 or 15, and at least 80%, preferably, at least 80% of the amino acid sequence of SEQ ID NO: 1 or 15 over the entire length thereof. Include polypeptides comprising an amino acid sequence having at least 90%, more preferably 95%, even more preferably at least 97-99% identity. It also has at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97-99% identity to either the amino acid sequence of SEQ ID NO: 1 or 15 over its entire length. Also included are polypeptides having the amino acid sequence. The polypeptide of the present invention further includes a polypeptide comprising an amino acid sequence encoded by a polynucleotide having at least 80% identity over its entire length to the nucleotide sequence of SEQ ID NO: 2 or 16. These polypeptides may be in the form of the "mature" protein or may be part of a larger protein, such as a fusion protein. It is often advantageous to include additional amino acid sequences, such as secretory or leader sequences, prosequences, sequences useful for purification such as multiple histidine residues, or There are additional sequences to ensure stability. Also, fragments of the polypeptide are included in the present invention. Such a fragment is a polypeptide having an amino acid sequence that entirely is the same as part, but not all, of the amino acid sequence of the polypeptide. Such fragments may be "free-standing" or contained within a larger polypeptide, i.e., a portion or region of the larger polypeptide, most preferably one contiguous sequence. The region may be composed of fragments thereof. Representative examples of polypeptide fragments of the present invention include, for example, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to SEQ ID NO: 1 or 15 And fragments up to the terminus. Here, “approximately” includes a range in which several, five, four, three, two, or one amino acid increases or decreases at one or both ends of the above range. Suitable fragments include, for example, deletions of a series of residues including the amino terminus or a series of residues including the carboxyl terminus, or deletion of a series of residues including the amino terminus and the carboxyl terminus. Excluding the loss, a truncation polypeptide having the amino acid sequence of SEQ ID NO: 1 or 15 is included. In addition, α helix and α helix forming region, β sheet and β sheet forming region, turn and turn forming region, coil and coil forming region, hydrophilic region, hydrophobic region, α amphiphilic region, β amphiphilic region, Also suitable are fragments characterized by structural or functional properties, such as those that include variable regions, surface-forming regions, substrate binding regions, and high antigen index regions. Other suitable fragments are biologically active fragments. Biologically active fragments are those that mediate a biological activity, such as an aspartic protease activity, including fragments with similar activity, increased activity, or reduced undesirable activity. . Also included are fragments that are antigenic or immunogenic in animals, particularly humans. Preferably, all of these polypeptide fragments retain the biological activity of the precursor polypeptide, including antigenic activity. Variants of the specified sequences and fragments also form part of the invention. Preferred variants are those that differ from the subject by conservative amino acid substitutions, i.e., those that substitute a residue with another having similar properties. Typical such substitutions are between Ala, Val, Leu and Ile; between Ser and Thr; between the acidic residues Asp and Glu; between Asn and Gln; between the basic residues Lys and Arg; Occurs between group residues Phe and Tyr. In particular, a mutant in which several, 5 to 10, 1 to 5 or 1 to 2 amino acids are substituted, deleted or added in any combination is preferable. The polypeptide of the present invention can be produced by any appropriate method. Such polypeptides include isolated natural polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. It is. Means for producing such polypeptides are well understood in the art. According to another aspect of the present invention, there is provided a polynucleotide (DNA or RNA) encoding the polypeptide. In a preferred embodiment of the present invention, there is provided a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 15. In particular, the present invention provides a polynucleotide having the DNA sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 16. The present invention further provides a polynucleotide encoding the polypeptide, comprising the DNA sequence shown in SEQ ID NO: 2 or SEQ ID NO: 16. The polynucleotide of the present invention may further comprise at least 80%, preferably at least 90%, more preferably at least 95%, over its entire length, of the nucleotide sequence encoding the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 15. At least 80%, preferably at least 90%, more preferably at least 95% identity over the entire length of a polynucleotide comprising a nucleotide sequence having identity and the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 16 And polynucleotides comprising the nucleotide sequence having the same. Further, those with at least 97% identity are more preferred, those with at least 98-99% identity are even more preferred, and those with at least 99% identity are most preferred. CDNA molecules (ESTs) exhibiting extensive identity to the cDNAs of SEQ ID NO: 2 and SEQ ID NO: 16 have been identified in human-derived cDNA libraries generated from various sources. These ESTs are shown in SEQ ID NOs: 3-14. SEQ ID NO: Library: SEQ ID NO: 3 (EST 176432) Raji cells, cyclohexamide treated SEQ ID NO: 4 (EST 424772) Human B cell lymphoma SEQ ID NO: 5 (EST 443275) Thoracic lymph node cDNA library SEQ ID NO: 6 (EST 567394) Raji cells, cyclohexamide treated SEQ ID NO: 7 (EST 685578) Human activated monocyte SEQ ID NO: 8 (EST 928138) Fetal liver, subtraction II SEQ ID NO: 9 (EST 947785) Thoracic lymph node cDNA library SEQ ID NO: 10 (EST 1000163) spinal cord SEQ ID NO: 11 (EST 1218021) spleen, chronic lymphocytic leukemia SEQ ID NO: 12 (EST 1320439) human tonsils, lib2 SEQ ID NO: 13 (EST 716478) cd34 depleted buffy coat cord blood SEQ ID NO: 14 (EST 857644) Human adult testis, large insert Accordingly, in a further aspect, the present invention relates to one or more partial DNAs selected from SEQ ID NOs: 3-14. Providing a polynucleotide encoding the aspartic protease characterized by columns. The polynucleotides of SEQ ID NO: 2 and SEQ ID NO: 16 are structurally related to the aspartic protease family. SEQ ID NO: 2 and SEQ ID NO: 16 have 1376 and 1347 nucleotides, respectively. The first (ie, N-terminal) active site motif is believed to be -Ala-Phe-Asp-Thr-Gly- and, due to at least two ESTs (SEQ ID NOs: 11 and 12), in each case the same DNA Encoded with the sequence (-GCC-TTT-GAC-ACT-GGC-). The second (ie, C-terminal) active site motif is believed to be -Ile-Leu-Asp-Thr-Gly-, and by at least one EST (SEQ ID NO: 13) (DNA sequence-ATC-CTG-GAT- ACA-GGC-). The conserved flap region of this enzyme is believed to be -Tyr-Gly-Thr-Gly-, and in all cases at least four ESTs (-TAT-GGA-ACT-GGG-) having the same DNA sequence (-TAT-GGA-ACT-GGG-) SEQ ID NOs: 9, 10, 5 and 12). The sequences of SEQ ID NO: 1 and SEQ ID NO: 15 are considered to include the full length of the mature form of the novel aspartic protease, plus about 60 amino acids containing the pro-portion (or pro-segment). The starting Met residue was also identified in SEQ ID NO: 1 and the ORF starts at nucleotide 26. Convenient blast sequence analysis shows that the polypeptide of SEQ ID NO: 15 shows the highest homology to chicken preprocathepsin D. However, at the functional level, the polypeptides of SEQ ID NO: 1 and SEQ ID NO: 15 have a cathepsin E-specific glycosylation site (-Asn-Phe-Thr-) immediately preceding the sequence encoding the N-terminal active site motif. It appears to be most similar to cathepsin E because of its presence. The wide distribution of the ESTs associated with this polypeptide in many cell types suggests that the encoded polypeptide has a non-specific hydrolytic function (similar to cathepsin E and D). . The polynucleotide of the present invention may be in the form of RNA or DNA, and DNA includes cDNA, genomic DNA and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or the non-coding (antisense) strand. The coding sequence encoding a polypeptide of the present invention may be identical to the coding sequence shown in SEQ ID NO: 2 or SEQ ID NO: 16 because of the redundancy or degeneracy of the genetic code. It may be a different coding sequence that encodes the same polypeptide as DNA. The present invention includes variants of the above polynucleotides that encode fragments, analogs and derivatives of the polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 15. A polynucleotide variant can be a naturally occurring allelic or non-naturally occurring variant of the polynucleotide. Thus, the present invention encompasses polynucleotides that encode the same polypeptide as the polypeptide set forth in SEQ ID NO: 1 or SEQ ID NO: 15, as well as variants of the polynucleotide that encode fragments, derivatives or analogs of the polypeptide. I do. Such nucleotide variants include deletion variants, substitution variants, and addition or insertion variants. A polynucleotide of the invention may have a coding sequence that is a natural allelic variant of the coding sequence of SEQ ID NO: 2 or SEQ ID NO: 16. As is known in the art, an allelic variant is another form of a polynucleotide sequence having one or more nucleotide substitutions, deletions or additions that does not substantially alter the function of the encoded polypeptide. is there. The polynucleotide encoding the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 15 comprises only the coding sequence of the polypeptide, the coding sequence of the polypeptide and an additional coding sequence such as a leader or secretory sequence or a proprotein sequence; It can include non-coding sequences such as non-coding sequences and introns 5 'and / or 3' to the coding sequence of the polypeptide (and optionally additional coding sequences) and the coding sequence of the mature polypeptide. Thus, "polynucleotide encoding a polypeptide" includes not only a polynucleotide comprising only the coding sequence of the polypeptide, but also a polynucleotide comprising additional coding and / or non-coding sequences. The invention therefore provides polynucleotide sequences whose coding sequence is useful for expression and secretion of the polypeptide from a host cell (eg, a leader sequence that functions as a secretory sequence that controls the transport of the polypeptide from the host cell). Comprises a polynucleotide fused in the same reading frame. A polypeptide having a leader sequence is a preprotein that, when cleaved by the host cell, produces a mature polypeptide. Such a polynucleotide can also encode a proprotein in which 5 'amino acid residues have been added to the mature protein. A mature protein having a prosequence is a proprotein and is an inactive form of the protein. Cleavage of this prosequence results in an active mature protein. Thus, for example, a polynucleotide of the invention may encode a mature protein, a protein having a prosequence, or a protein having both a prosequence and a presequence (leader sequence). A polynucleotide of the invention may also have the coding sequence fused in the same reading frame to a marker sequence that allows for purification of the polypeptide of the invention. The marker sequence may be a hexa-histidine tag supplied by the pQE-9 vector, which in the case of a bacterial host allows for purification of the mature polypeptide fused to the marker, and may include, for example, a mammalian host (eg, : COS-7 cells), the marker sequence may be a hemagglutinin (HA) tag. This HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I. et al. Cell, 37: 767 (1984)). The present invention further relates to polynucleotides that hybridize to said sequences when there is at least 50%, preferably 70%, identity between the sequences. The invention particularly relates to polynucleotides that hybridize under stringent conditions to the above polynucleotides. As used herein, "stringent conditions" refers to conditions under which hybridization will occur only when there is at least 95%, preferably at least 97%, identity between the sequences. In a preferred embodiment, the polynucleotide that hybridizes to the above polynucleotide encodes a polypeptide that retains substantially the same biological function or activity as the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 15. The terms "fragment,""derivative," and "analog" when referring to a polypeptide of SEQ ID NO: 1 or SEQ ID NO: 15 retain essentially the same biological function or activity as such polypeptide Means a polypeptide. Thus, analogs include proproteins that are activated by cleavage of the proprotein portion to produce a mature form of the active polypeptide. As used herein, the term "identity" is a measure of the identity of nucleotide or amino acid sequences. Generally, sequences are aligned so as to provide the greatest match. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. For example, COMPUTATIONALM OLECULAR BIOLOGY, Lesk, A. M. Ed., Oxford University Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W. Ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PARTI, Griffin, A. M. and Griffin, H. G. Ed., Humana Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G. Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER, Gribskov, M.S. and Devereux, J. Ed., M Stockton Press, New York, 1991. While there are many ways to determine the identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to those of skill in the art (Carillo, H. et al. and Lipton, D. , SIAM J Applied Math (1988) 48: 1073). Commonly used methods for determining the identity or similarity between two sequences include Guide to Huge Computers, Martin J. Bishop, Academic Press, San Diego, 1994 and Carillo, H .; and Lipton, D. , SIAM J Applied Math (1988) 48: 1073, but is not limited thereto. Methods for determining identity and similarity are codified in computer programs. Suitable computer programming methods for determining identity and similarity between two sequences include the GCS program package (Devereux, J .; Et al., Nucleic Acids Research (1984) 12 (1): 387), BLASTP, BLASTN, FASTA (Atschul, S. F. J Molec Biol (1990) 215: 403), but are not limited thereto. By way of example, a polynucleotide having a nucleotide sequence that has, for example, at least 95% "identity" to a reference nucleotide sequence of SEQ ID NO: 2/16 is defined as a polynucleotide having a nucleotide sequence of SEQ ID NO: 2/16 It is intended to be identical to the reference sequence except that it can contain up to 5 point mutations for each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted, replaced with other nucleotides, or Up to 5% of the total number of nucleotides may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5 'or 3' terminal positions of the reference nucleotide sequence, or anywhere between these terminal positions, individually between nucleotides in the reference sequence or within the reference sequence. Can be arranged as one or more contiguous groups. Similarly, for example, a polypeptide having an amino acid sequence having "identity" of at least 95% with respect to the reference amino acid sequence of SEQ ID NO: 1/15 is defined as having an amino acid sequence of this polypeptide which is 100% It is intended to be identical to the reference sequence except that it can include up to 5 amino acid changes per amino acid. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted, replaced with other amino acids, or The number of amino acids up to 5% of all amino acid residues in the reference sequence may be inserted into the reference sequence. These changes in the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence, or anywhere between these terminal positions, individually between amino acid residues in the reference sequence, or within the reference sequence. It can be arranged as one or more consecutive groups. The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, but is preferably a recombinant polypeptide. Fragments, derivatives or analogs of the polypeptides of SEQ ID NO: 1 or SEQ ID NO: 15 are those wherein (i) one or more amino acid residues are replaced with conservative or non-conservative amino acid residues (preferably conservative amino acid residues) (Substituted amino acid residues may or may not be encoded in the genetic code), (ii) one or more amino acid residues include a substituent, (iii) the mature polypeptide Those fused to other compounds, such as compounds that increase the half-life of the polypeptide (eg, polyethylene glycol), or (iv) additional amino acids (eg, leader or secretory sequence, mature polypeptide) to the mature polypeptide Or the sequence used for purification of the protein or the proprotein sequence). Such fragments, derivatives and analogs are considered to be within the skill of those in the art from the teachings herein. The polypeptides and polynucleotides of the present invention are preferably obtained in an isolated form, and are advantageously purified to homogeneity. The term "isolated" means that the substance has been separated from its original environment (eg, the natural environment if it occurs naturally). For example, a natural polynucleotide or polypeptide present in a living animal has not been isolated, but a polynucleotide or polypeptide isolated from some or all of the coexisting substances in the natural system has been isolated. is there. Such polynucleotides may be part of a vector, and / or such polynucleotides or polypeptides may be part of a composition, and such vectors or compositions may be part of their natural environment. It is also isolated in that it is not a part. Preferably, the polypeptide is in purified form. Purified form means at least 80%, preferably 90%, more preferably 95%, and most preferably 99%, purified relative to other protein contaminants. The DNA of the present invention also allows animals that cannot or do not express this enzyme to be developed by homologous recombination or "knockout" strategies (Kapecchi, Science, 244: 1288-1292 (1989)). The present invention further relates to a vector containing the polynucleotide of the present invention, a host cell genetically engineered using the vector of the present invention, and the production of the polypeptide of the present invention by a recombinant method. Thus, according to yet another aspect of the present invention, a recombinantly produced polypeptide of the present invention comprising expressing a polynucleotide encoding the polypeptide of the present invention in a host and recovering the expression product. A production method is provided. Alternatively, the polypeptide of the present invention can be produced synthetically using a conventional peptide synthesizer. The host cell is genetically engineered (transduced, transformed or transfected) with a vector of the invention, which can be, for example, a cloning vector or an expression vector. This vector may be in the form of, for example, a plasmid, cosmid, phage and the like. The genetically engineered host cells are cultured in a conventional nutrient medium, which is suitably modified for promoter activation, transformant selection, or gene amplification. Culture conditions, such as temperature, pH, etc., are the same as those previously used for the host cell selected for expression and will be apparent to those skilled in the art. Suitable expression vectors include chromosomal, non-chromosomal and synthetic DNA sequences (eg, SV40 derivatives), bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmid and phage DNA, viral DNA ( Examples: vaccinia, adenovirus, fowlpox virus, pseudorabies virus). However, other vectors can be used as long as they can replicate and survive in the host. Various methods can be used to insert the appropriate DNA sequence into the vector. Generally, the DNA sequence is inserted into an appropriate restriction endonuclease site by methods known in the art. These and other methods are deemed to be within the skill of those in the art. The DNA sequence in the expression vector is operably linked to a suitable expression control sequence (promoter) that directs the synthesis of mRNA. The following can be mentioned as typical examples of such promoters. That is, LTR or SV40 promoter, E. coli lac or trp, phage λP _L Promoters and other promoters known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator, and may contain appropriate sequences for expression amplification. In addition, the expression vector may have one or more selectable marker genes that confer phenotypic properties for selecting transformed host cells, for example, dihydrofolate reductase or neomycin resistance in eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli. It is preferred to include. The gene is placed under the control of a promoter, a ribosome binding site (for bacterial expression), and optionally an operator (collectively referred to herein as "regulatory" elements), and thus contains the expression construct. In a host cell transformed with the vector, a DNA sequence encoding the desired protein is transcribed into RNA. A coding sequence may or may not include a signal peptide or leader sequence. The protein sequences of the present invention can be expressed using, for example, the E. coli tac promoter or the protein A gene (spa) promoter and signal sequence. The leader sequence is removed by the bacterial host during post-translational processing. The promoter region can be selected from the desired gene using a CAT (chloramphenicol transferase) vector or other vector with a selectable marker. Two suitable vectors are PKK232-8 and PCM7. Particularly, bacterial promoters include lacI, lacZ, T3, T7, gpt, λP _R , P _L And trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus and mouse metallothionein-I. Selection of the appropriate vector and promoter is within the skill of the art. In addition to regulatory sequences, it may be desirable to add regulatory sequences that can regulate the expression of the protein sequence against the growth of the host cell. Regulatory sequences are known to those of skill in the art, and include, for example, those that induce or discontinue gene expression in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements, such as enhancer sequences, may be present in the vector. Expression vectors are constructed to position a particular coding sequence in a vector with the appropriate regulatory sequences, and the positioning and orientation of the coding sequence relative to the control sequences is such that the coding sequence is transcribed under "control" of the control sequences ( That is, the RNA polymerase that binds to the DNA molecule at the position of the control sequence transcribes the coding sequence). It may be desirable to modify the coding sequence to achieve this purpose. For example, in some cases, it is necessary to modify the coding sequence so that the coding sequence can be joined to the control sequence in the proper orientation, ie, maintain the reading frame. The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector (eg, the cloning vector described above). Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and appropriate restriction sites. In addition, to enhance expression, the coding sequence may be modified to use codons compatible with the given host cell. Generally, recombinant expression vectors contain an origin of replication, a selectable marker that allows for the transformation of host cells (eg, the ampicillin resistance gene of E. coli and the TRP1 gene of S. cerevis iae), and the transcription of downstream structural sequences. Would include a promoter from a highly expressed gene. The heterologous structural sequence is assembled in proper reading frame with the translation initiation and termination sequence and, preferably, a leader sequence capable of directing secretion of the translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence may encode a fusion protein containing an N-terminal identifying peptide that confers the desired property (eg, stabilization or convenient purification of the recombinant expression product). An appropriate host can be transformed with a vector containing the appropriate DNA sequence and an appropriate promoter or control sequence as described above, and the protein can be expressed in the host. Examples of recombinant DNA vectors for cloning and host cells which can be transformed are bacteriophage λ (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (Gram negative bacteria), pGV1106 (Gram negative bacteria). , PLAFR1 (Gram-negative bacteria), pME290 (Non-E. Coli Gram-negative bacteria), pHV14 (Escherichia coli and Bacillus subtilis), pBD9 (Bacillus subtilis), pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), baculo A viral insect cell line, such as YCp19 (Saccharomyces). For a review, see “DNA Cloning”: Vols. I & II, edited by Glover et al., IRL Press Oxford (1985) (1987) and See Maniatis et al., "Molecular Cloning" Cold Spring Harbor Laboratory (1982). In some cases, it may be desirable to add a sequence that causes secretion of the polypeptide from the host organism and subsequent cleavage of the secretory signal. Yeast expression vectors are also known in the art. See, for example, U.S. Patent Nos. 4,446,235, 4,443,539, and 4,430,428. See also European Patent Applications 103,409, 100,561, 96,491. pSV2 _neo (Described in J. Mol. Appl. Genet. 1: 327-341), which uses the SV40 late promoter to direct expression in mammalian cells and uses the vector pCDNA1 derived from pCDNA1. _neo (Mol. Cell Biol. 7: 4125-29) use the CMV promoter to direct expression. Both of these two types of vectors are used for transient or stable (using G418 resistance) expression in mammalian cells. Insect cell expression systems (eg, Drosophila) are also useful; see, for example, PCT applications WO 90/06358 and WO 92/06212 and EP290,261-B1. The polypeptide can be expressed in a host cell under the control of a suitable promoter. Cell-free translation systems can also be used to produce the proteins using RNA derived from the DNA constructs of the present invention. Cloning and expression vectors suitable for use with prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NJ. Y. (1989), the disclosure of which is incorporated herein by reference. Transcription of a DNA encoding a polypeptide of the present invention by higher eukaryotic cells can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 bp, that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin from bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. In a further aspect, the present invention relates to a host cell comprising the above-described vector. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, and the host cell can be a prokaryotic cell, such as a bacterial cell. The following may be mentioned as typical examples of suitable hosts. As prokaryotic cells, for example, Escherichia coli, Streptomyces, bacterial cells such as Salmonella atyphimurium, as eukaryotic cells, for example, fungal cells such as yeast, Drosophila (Drosophila) and Spodoptera frugiperda (Spodopteraf rugiperda) ), Mammalian cells such as CHO, COS or Bowes melanoma, plant cells and the like. The selection of a suitable host is considered to be within the skill of those in the art from the teachings herein. Introduction of the construct into host cells can be performed by calcium phosphate transfection, DEAE-dextran mediated transfection, electroporation, etc. (Davis, L. et al.). , Dibner, M. , Batty, I. , Basic Methods in Molecular Biology (1986)). After transformation of the appropriate host strain and propagation of the host strain to an appropriate cell density, the given promoter is induced by appropriate means (eg, temperature shift or chemical induction) and the cells are cultured for an additional hour. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is saved for subsequent purification. The microbial cells used for protein expression can be disrupted by any method, including freeze-thaw cycling, sonication, mechanical disruption or the use of cell lysing agents, and such methods are known to those skilled in the art. It is. Various mammalian cell culture systems can also be used to express the recombinant protein. Examples of mammalian expression systems include the COS-7 line of monkey kidney fibroblasts described in Gluzman, Cell, 23: 175 (1981), and other cell lines that can express compatible vectors, such as , C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will contain an origin of replication, a suitable promoter and enhancer, and will also contain necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5 'flanking untranslated sequences. . DNA sequences from the SV40 splice and polyadenylation sites may be used to obtain the required non-transcribed genetic elements. The polypeptide of the present invention is produced by growing a host cell transformed with the above-mentioned expression vector under the conditions under which the desired polypeptide is expressed, depending on the selected expression system and host. The polypeptide is then isolated from the host cell and purified. If the expression system secretes the polypeptide into a growth medium, the polypeptide can be purified directly from the medium. If the polypeptide is not secreted, it is isolated from the cell lysate or recovered from the cell membrane fraction. Where the polypeptide is localized on the cell surface, whole cells or discrete membranes can be used as an assayable source of the desired gene product. Polypeptides expressed by bacterial hosts such as E. coli may require isolation from inclusion bodies and regeneration / reconstitution. Selection of the appropriate growth conditions and recovery methods are within the skill of the art. Polypeptides can be recovered and purified from recombinant cell culture by ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography. Various methods can be used, including chromatography, and lectin chromatography. Optionally, a protein regeneration step may be employed to complete the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be used as a final purification step. Depending on the host used in the recombinant production method, the polypeptide of the present invention may be glycosylated or non-glycosylated. Also, the polypeptide of the present invention may include the first methionine amino acid residue. The polypeptides of the present invention are also useful in identifying other molecules with similar biological activity. An example of such a screen is to isolate the coding region of the aspartic protease gene, either by synthesizing an oligonucleotide probe or by using a DNA sequence known as the probe itself. Using a labeled oligonucleotide having a sequence complementary to the gene sequence of the present invention, a library of human cDNA, genomic DNA or mRNA is screened to determine with which member of the library the probe hybridizes. The polypeptides of the present invention can also be used according to the present invention by in vivo expression of the polypeptide, often referred to as "gene therapy". Thus, for example, cells from a patient are engineered ex vivo with a polynucleotide (DNA or RNA) encoding a polypeptide, and the engineered cells are then introduced into a patient to be treated with the polypeptide. I do. Such methods are known in the art. For example, cells can be genetically engineered by known methods by using retroviral particles containing RNA encoding the polypeptide of the present invention. Similarly, cells can be engineered in vivo for expression of a polypeptide in vivo, for example, by methods known in the art. As is known in the art, producer cells for producing retroviral particles containing RNA encoding a polypeptide of the present invention can be used to engineer cells in vivo and to express polypeptides in vivo. May be administered to patients. Methods of administering the polypeptides of the present invention and other methods by such methods will be apparent to those skilled in the art from the teachings of the present invention. For example, an expression vehicle for genetically engineering cells may be other than a retrovirus, such as an adenovirus, for genetically engineering cells in vivo after combining an adenovirus with a suitable delivery vehicle. Can be used for A “recombinant” polypeptide is a polypeptide produced by recombinant DNA methods, ie, a polypeptide produced from a cell transformed with a foreign DNA construct encoding a polypeptide of interest. "Synthetic" polypeptides are those produced by chemical synthesis. A "replicon" is a genetic element (eg, a plasmid, chromosome, virus) that functions as an autonomous DNA replication unit in vivo, ie, capable of replicating under its own control. A "vector" is a replicon, such as a plasmid, phage, cosmid, or the like, that can ligate to another DNA segment and cause replication of the ligated segment. "Double-stranded DNA molecule" refers to a polymeric form of deoxyribonucleotides (bases adenine, guanine, thymine or cytosine) in the form of a double-stranded helix (both relaxed and supercoiled). The term refers only to the primary and secondary structure of the molecule and is not meant to limit it to a particular tertiary form. Thus, the term includes, among others, linear DNA molecules (eg, restriction fragments), viruses, plasmids, and double-stranded DNA present in chromosomes. In describing the structure of a particular double-stranded DNA molecule, the sequences are described herein according to the convention shown in the 5 'to 3' direction along the sense strand of DNA. The DNA "coding sequence" of a particular protein or the "nucleotide sequence encoding a particular protein" refers to a DNA sequence that is transcribed and translated into a polypeptide when placed under the control of appropriate regulatory sequences. A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 'direction) coding sequence. The transcription initiation site (conveniently defined by mapping with nuclease S1) and the protein binding domain (consensus sequence) involved in binding RNA polymerase will be found in the promoter sequence. Eukaryotic promoters will often (but not always) include a "TATA" box and a "CAT" box. A DNA "control sequence" is a collection of promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, that provide for the expression (ie, transcription and translation) of a coding sequence in a host cell. It is a term that points to. The control sequence directs “expression” of the coding sequence in the cell when the RNA polymerase binds to the promoter sequence and transcribes the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence. ". A “host cell” is a cell that has been transformed or transfected with a foreign DNA sequence, or is capable of transformation or transfection with a foreign DNA sequence. A cell has been "transformed" by exogenous DNA when the exogenous DNA has been introduced inside the cell membrane. The foreign DNA may or may not be integrated (covalently linked) into the chromosomal DNA making up the genome of the cell. For example, in prokaryotes and yeast, foreign DNA may be retained on episomal elements such as plasmids. With respect to eukaryotic cells, a stably transformed or transfected cell is one in which foreign DNA has been integrated into the chromosome so that it may be inherited by daughter cells through chromosomal replication. This stability is demonstrated by the ability of eukaryotic cells to establish cell lines or clones consisting of a population of daughter cells containing foreign DNA. A "clone" is a population of cells derived from a single cell or a common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations. Two DNA or polypeptide sequences are "substantially" when at least about 85% (preferably at least about 90%, most preferably at least about 95%) of their nucleotides or amino acids are matched over a length of the molecule. Are "homologous to" or "substantially identical" and include allelic variants. "Substantially homologous," as used herein, also refers to sequences that show identity to a particular DNA or polypeptide sequence. A substantially homologous DNA sequence can be identified by Southern hybridization experiments under conditions (eg, stringent) defined for the particular system. Defining appropriate hybridization conditions is within the skill of the art. For example, “Current Protocols in Mol. Biol. ”Vol. See I & II, Ed., Wiley Interscience, Ausbel et al. (1992). Protein sequences that are substantially identical can be identified by proteolytic digestion, gel electrophoresis, and microsequencing. The term "functionally equivalent" means that the amino acid sequence of the protein of interest exhibits the same type of enzymatic activity as the aspartic protease. A "heterologous" region of a DNA construct is an identifiable DNA segment within or attached to another DNA molecule that is not naturally associated with another molecule. The polypeptides of the present invention can be used for therapy. Thus, in a further aspect, the present invention provides a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 15, and fragments, analogs or derivatives thereof, for use in therapy. Suitably, such polypeptides may play a role in preventing, ameliorating and treating dysfunctions or diseases including but not limited to hypertension, inflammation, asthma and cardiopulmonary disease. The polypeptides and polynucleotides of the present invention are used with suitable pharmaceutical carriers. Such compositions will contain a therapeutically effective amount of the active agent and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration. The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. Such containers include a notice in the form of a government agency that regulates the manufacture, use, or sale of pharmaceuticals or biological products, which is approved by a government agency for manufacture, use, or sale for human administration. ) Can be attached. Further, the polypeptides of the present invention may be used with other therapeutic compounds. The pharmaceutical compositions can be administered in a convenient manner, for example, orally, topically, intravenously, intraperitoneally, intramuscularly, subcutaneously, intranasally or intradermally. The polypeptide or polynucleotide of the invention is administered in an amount effective to treat and / or prevent a particular indication. The amount and dosage regimen of the active agent administered to a subject will depend on many factors, such as the mode of administration, the nature of the condition to be treated, the judgment of the practitioner, and the like. The sequences of the present invention are also useful for chromosome identification. Such sequences can be specifically targeted to and hybridized to specific locations on individual human chromosomes. In addition, there is an ongoing need to identify specific sites on chromosomes. Currently, chromosome marking reagents based on actual sequence data (repeated polymorphisms) are available to indicate chromosomal location. Mapping DNA to chromosomes according to the present invention is an important first step in correlating such sequences with disease-related genes. Briefly, the sequence can be mapped to a chromosome by generating PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of cDNA is used to quickly select primers that do not span more than one exon in genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment. PCR mapping of somatic cell hybrids is a rapid method for assigning specific DNA to specific chromosomes. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved in a similar manner using a panel of fragments from a particular chromosome or a pool of large genomic clones. Other mapping strategies that can also be used to map to chromosomes include in situ hybridization, prescreening using labeled flow sorted chromosomes, and preselection by hybridization to construct chromosome-specific cDNA libraries. It is. Fluorescent in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal region can be used to obtain a precise chromosomal location in one step. This technique can be used for cDNAs as short as 500 or 600 bases. However, clones larger than 2,000 bp are more likely to bind to a unique chromosomal location with sufficient signal intensity for convenient detection. FISH requires the use of clones (from which the ESTs were derived), the longer the better. For example, 2,000 bp is better and 4,000 is better. Probably not more than 4,000 is needed to get good results (reasonable percentage of time). For a discussion of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergam on Press, New York (1988). Once a sequence has been mapped to a precise chromosomal location, the physical location of the sequence on this chromosome can be correlated with genetic map data. This kind of data is for example V. McKusick, found in Mendelian Inheritance in Man (available online at Johns Hopkins University Welch Medical Library). Thereafter, the relationship between the disease and the gene mapped to the same chromosomal region is confirmed by linkage analysis (co-inheritance of physically adjacent genes). Next, it is necessary to examine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If the mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation may be responsible for the disease. According to the current resolution of physical and genetic mapping techniques, a cDNA that is correctly located in a chromosomal region associated with a disease will be one of 50-500 possible causative genes (one of Assume megabase mapping resolution and 1 gene / 20 kb). Comparison of affected and unaffected individuals generally involves first looking for structural changes in the chromosome, such as deletions or translocations, that are visible from the chromosome spread or detectable by PCR based on the cDNA sequence. including. Ultimately, complete sequencing of genes from several individuals is required to confirm the presence of the mutation and distinguish between the mutation and the polymorphism. The polypeptides of the present invention or cells expressing them can be used as an immunogen to produce antibodies against them. These antibodies can be, for example, polyclonal or monoclonal antibodies. The invention also includes chimeric antibodies, single-chain antibodies, humanized antibodies, and Fab fragments, or the products of a Fab expression library. Various methods known in the art can be used for producing such antibodies and fragments. Antibodies to the polypeptide of the present invention can be obtained by directly injecting the polypeptide into an animal, or by administering the polypeptide to an animal (preferably other than a human). The antibody thus obtained will bind to the polypeptide itself. In this way, antibodies can be generated that bind to the complete native polypeptide, even when sequences encoding fragments of the polypeptide are used. Thereafter, using such an antibody, the polypeptide can be isolated from a tissue expressing the polypeptide. To make monoclonal antibodies, any technique which provides antibodies produced from continuous cell line cultures can be used. Examples include the hybridoma method (Kohler and Milstein, 1975, Nature, 256: 495-497), the trioma method, the human B-cell hybridoma method (Kozbor et al., 1983, Immunology Today 4:72), and human monoclonal antibodies. EBV-hybridoma method for production (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. , Pp. 77-96). The techniques described for generating single-chain antibodies (US Pat. No. 4,946,778) can also be applied to generate single-chain antibodies to the immunogenic polypeptide products of the invention. The invention further provides a compound screening method for identifying a compound that inhibits the polypeptide, the method comprising contacting the isolated polypeptide with a test compound and determining a turnover rate in the absence of the test compound. Measuring the rate of turnover of the enzyme substrate as compared to. The invention also relates to the compounds identified thereby. The present invention also provides a transgenic non-human animal containing a polynucleotide encoding the polypeptide of the present invention. Also provided are methods for using the transgenic animals described above as models for mutation and SAR (structure / activity relationship) evaluation or in drug screening. The invention also relates to inhibitor molecules of the polypeptides of the invention and their use in reducing or eliminating the function of the polypeptide. An example of an inhibitor is an antibody, and in some cases, an oligonucleotide that binds to the polypeptide. An example of an inhibitor is an antisense construct made using antisense technology. Using antisense technology, gene expression can be controlled by triple helix formation or antisense DNA or RNA, both of which methods are based on binding of the polynucleotide to DNA or RNA. For example, the 5 'coding portion of a polynucleotide sequence encoding a polypeptide of the invention can be used to design antisense RNA oligonucleotides about 10-40 base pairs in length. DNA oligonucleotides are designed to be complementary to the region of the gene involved in transcription (triple helix-Lee et al., Nucl. Acids Res. Sci., 6: 3073 (1979); Cooney et al., Science, 241: 456 (1988); and Dervan et al., Science, 251: 1360 (1991)), which prevents transcription and production of the polypeptide. Antisense RNA oligonucleotides hybridize to mRNA in vivo and block translation of the mRNA molecule into a polypeptide (Okano, J. et al. Neurochem. , 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can also be delivered to cells to express antisense RNA or DNA in vivo, thereby suppressing polypeptide production. Another example of an inhibitor is a small molecule that binds to and occupies the catalytic site of a polypeptide, which renders the catalytic site inaccessible to the substrate, resulting in normal biological activity. Is disturbed. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules. When used in therapy, the inhibitors of the invention are formulated according to standard pharmaceutical practice. Inhibitors that are active when administered orally can be formulated as solutions, for example, syrups, suspensions or emulsions, tablets, capsules and lozenges. Liquid formulations generally are suspended or dissolved in a suitable liquid carrier such as ethanol, glycerin, non-aqueous solvents (eg, polyethylene glycol), oils, water and the like, including suspending agents, preservatives, flavoring or coloring agents. It consists of a suspension or solution of the compound or a pharmaceutically acceptable salt thereof. A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier (s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose. A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, a pellet containing the active ingredient may be prepared using a standard carrier and then filled into a hard gelatin capsule with the pellet, or alternatively, the dispersion or suspension may be prepared with a suitable pharmaceutical carrier (e.g., , Aqueous gums, celluloses, silicates, oils), and then the dispersion or suspension is filled into soft gelatin capsules. A typical parenteral composition is a compound or a pharmaceutical composition thereof dissolved or suspended in a sterile aqueous carrier or parenterally acceptable oil (eg, polyethylene glycol, polyvinylpyrrolidone, lecithin, peanut oil, sesame oil). Consisting of a salt acceptable to Alternatively, the solution can be lyophilized and reconstituted with a suitable solvent just prior to administration. A typical suppository is a compound of formula (I) or a pharmaceutically acceptable salt thereof, which is active when administered in this manner, with a binder and / or lubricant (eg, polymeric glycols, gelatin, cocoa butter), Or with other low melting point vegetable or synthetic waxes or fats. Preferably, the composition is in unit dosage form, such as a tablet or capsule. Each dosage unit for oral administration is preferably 1 to 250 mg (for parenteral administration, preferably 0.1 to 250 mg). 1-25 mg) of the inhibitor of the invention. The daily dosage regimen for adult patients may be, for example, an oral dose of 1-500 mg, preferably 1-250 mg, an intravenous, subcutaneous or intramuscular dose of 0. 1-100 mg, preferably 0. It may be 1 to 25 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof (calculated as the free base), wherein the compound is administered one to four times a day. Suitably, the compound will be administered over a continuous treatment period. The invention will be further described with reference to the following examples. However, it should be understood that the invention is not limited to such an embodiment. All parts or amounts are by weight unless otherwise specified. To facilitate understanding of the following examples, some commonly used methods and / or terms will be described. "Plasmids" are designated by a lower case letter preceded and / or followed by capital letters and / or numbers. The starting plasmids herein are commercially available, are available without limitation, or can be constructed from available plasmids according to published methods. In addition, plasmids equivalent to those described are known in the art and will be apparent to those skilled in the art. "Oligonucleotide" refers to a single-stranded polydeoxynucleotide or two complementary polydeoxynucleotide chains that can be chemically synthesized. Such synthetic oligonucleotides do not have a 5 'phosphate and thus are not ligated to other oligonucleotides without adding a phosphate group with ATP in the presence of a kinase. Synthetic oligonucleotides will be ligated to fragments that have not been dephosphorylated. "Ligation" refers to the method of forming a phosphodiester bond between two double-stranded nucleic acid fragments (Maniatis, T. et al. Id. , P. 146). Unless otherwise indicated, ligation reactions were performed with approximately equimolar amounts of DNA fragments to be ligated. The reaction is carried out using 10 units of T4 DNA ligase (ligase) per 5 μg under known buffer conditions. Sequence data SEQ ID NO: 1 SEQ ID NO: 2 c / a = This position is either C or A, now the unknown start ATG is underlined and the stop codon is unknown SEQ ID NO: 3 (EST 176432) SEQ ID NO: 4 (EST 424772) SEQ ID NO: 5 (EST 443275) SEQ ID NO: 6 (EST 567394) SEQ ID NO: 7 (EST 685578) SEQ ID NO: 8 (EST 928138) SEQ ID NO: 9 (EST 947785) SEQ ID NO: 10 (EST 1000163) SEQ ID NO: 11 (EST 1218021) SEQ ID NO: 12 (EST 1320439) SEQ ID NO: 13 (EST 716478) SEQ ID NO: 14 (EST 857644) SEQ ID NO: 15 446 amino acids (+3 additional stop codons) SEQ ID NO: 16

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 35/76 A61K 35/76 38/46 39/395 D 39/395 N 48/00 48/00 C07K 14 / 47 C07K 14/47 16/40 16/40 C12N 9/50 C12N 9/50 C12P 21/02 C C12P 21/02 C12Q 1/37 C12Q 1/37 A61K 37/54 (72) Inventor Powell, David United States of America 19426-0989 P. Collegeville, PA. Oh. Box 5089, South Collegeville Road 1250, Smith Kline Beechham Pharmaceuticals (72) Inventor Kay, John UK CEF1-3 TE Cardiff, University of Wales (72) Inventor Hill, Jeffrey CEF UK 13 TEE Cardiff, University of Wales

Claims (1)

[Claims] 1. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 15, or A nucleotide sequence encoding a fragment, analog or derivative of a polypeptide. To obtain a nucleotide sequence having at least 80% identity over its entire length. An isolated polynucleotide comprising: 2. A nucleotide comprising the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 16; The polynucleotide according to claim 1, which has an otide sequence. 3. For the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 16, Or a nucleotide sequence comprising at least 80% identity Polynucleotide. 4. The polynucleotide according to claim 3, which is the polynucleotide of SEQ ID NO: 1. . 5. 5. The method according to claim 1, wherein the polynucleotide is DNA. Polynucleotides listed. 6. 5. The method according to claim 1, wherein the polynucleotide is RNA. Polynucleotides listed. 7. The polynucleotide according to claim 5, wherein the polynucleotide is genomic DNA. Leotide. 8. A vector containing the DNA according to any one of claims 2, 4, 5, 6, and 7. Doctor. 9. A host cell genetically engineered with the vector of claim 8. Ten. A polypeptide encoded by the DNA from the host cell of claim 9. A method for producing a polypeptide, comprising expressing 11． 9. Manipulating cells with the vector of claim 8 by genetic engineering. A method for producing a cell capable of expressing a polypeptide. 12． Hybridizable to the polynucleotide according to any one of claims 1 to 7. And substantially the same biology as the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 15 A polynucleotide encoding a polypeptide having a specific function or activity. 13. Over the entire length of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 15 , Polypeptides having at least 80% identity, or fragments, analogs or Is a derivative. 14. A polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 15, Is a fragment, analog or derivative thereof. 15． The polypeptide has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 15 15. The polypeptide of claim 14, wherein 16． 16. The polypeptide according to claim 13, 14 or 15, which is in an isolated form. 17． 17. A polypeptide according to any one of claims 13 to 16 for use in therapy. 18． A method for identifying a compound that inhibits the polypeptide according to claim 13, 14, or 15. A method for screening a compound for isolating a polypeptide from a test compound. And measure the rate of turnover of the enzyme substrate to determine the rate in the absence of the test compound. A method comprising comparing with the rotation speed. 19. A compound identified by the method of claim 18. 20. An inhibitor of the polypeptide according to claim 13, 14 or 15. twenty one. 20.An antibody against the polypeptide according to claim 13, 14 or 15. The inhibitor according to any one of the above. twenty two. The polynucleotide according to claim 1 or 12, and the polynucleotide according to claim 13 or 14. A peptide, a compound according to claim 19, or an inhibitor and a product according to claim 20. A pharmaceutical composition comprising a pharmaceutically acceptable carrier. twenty three. Treatment of a patient in need of inhibiting the polypeptide according to claim 13, 14 or 15. 20. A method of treatment, wherein the patient is provided with a therapeutically effective amount of a compound or a therapeutic agent according to claim 19. 21. A method comprising administering the inhibitor of claim 20. twenty four. A compound according to claim 19 or claim 20 for producing a medicament for use in therapy. Use of the inhibitor according to the above.