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United States Patent RE43,166
Sato ,   et al. February 7, 2012

Gene encoding chondroitinase ABC and uses therefor

Abstract

Nucleic acid sequences coding for the chondroitinase ABC gene and isolated chondroitinase ABE protein produced in a host cell transformed with a nucleic acid vector directing the expression of a nucleotide sequence coding for chondroitinase ABE protein described. Chondroitinase ABC prepared by chemical synthesis also described. Monoclonal and polyclonal antibodies which are specifically reactive with chondroitinase ABC protein are disclosed. The isolated chondroitinase ABC can be used in methods of treating intervertebral disc replacement, promoting neurite regeneration, and detecting galactosaminoglycans.


Inventors: Sato; Nobuyuki (Ibaraki, JP), Shimada; Masahiko (Ibaraki, JP), Oda; Hiroshi (Ibaraki, JP)
Assignee: Maruha Nichiro Foods, Inc. (Tokyo, JP)
Appl. No.: 12/613,119
Filed: November 5, 2009


Related U.S. Patent Documents

Application NumberFiling DatePatent NumberIssue Date
08184435Jan., 1994
08074349Jun., 1993
Reissue of:08488960Jun., 19957008783Mar., 2006

Foreign Application Priority Data

Sep 22, 1992 [JP] 4-253016
Feb 24, 1993 [JP] 5-35810

Current U.S. Class: 435/232 ; 435/252.3; 435/252.33; 435/254.11; 435/320.1; 435/325; 435/69.1; 536/23.2
Current International Class: C12N 15/60 (20060101); C12N 5/10 (20060101); C12N 1/00 (20060101); C12N 1/21 (20060101); C12N 15/63 (20060101); C12N 9/88 (20060101)

References Cited

U.S. Patent Documents
4652639 March 1987 Stabinsky
4695816 September 1987 Essig et al.
4696816 September 1987 Brown
5198355 March 1993 Kikuchi et al.
5292509 March 1994 Hageman
Foreign Patent Documents
0 355 831 Feb., 1990 EP
0355831 Feb., 1990 EP
0-576-294 Dec., 1993 EP
0576294 Dec., 1993 EP
91/06303 May., 1991 WO
91/16070 Oct., 1991 WO
WO 91/16070 Oct., 1991 WO

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Primary Examiner: Prouty; Rebecca
Attorney, Agent or Firm: Nelson Mullins Riley & Scarborough LLP Remillard, Esq.; Jane E. Sloper, Esq.; Jill Gorny

Parent Case Text



This application is a continuation of application Ser. No. 08/184,435 filed on Jan. 14, 1994 now abandoned Entitled: Gene Encoding Chondroitinase ABC And Uses Therefor, which is a divisional of Ser. No. 08/074,349 filed Jun. 8, 1993 now abandoned.
Claims



What is claimed is:

.[.1. An isolated nucleic acid fragment encoding chondroitinase ABC, comprising the nucleotide sequence of SEQ ID NO: 1..].

.[.2. An expression vector comprising the nucleic acid as defined in claim 1 operably linked to a regulatory sequence..].

.[.3. A host cell transformed with the expression vector as defined in claim 2..].

4. A host cell of claim .[.3.]. .Iadd.23 .Iaddend.wherein the cell is eukaryotic.

5. A host cell of claim .[.3.]. .Iadd.23 .Iaddend.wherein the cell is prokaryotic.

.[.6. A method of producing chondroitinase ABC protein comprising: culturing the host cell as defined in claim 2 under conditions appropriate for expression; and isolating chondroitinase ABC protein from the culture..].

.[.7. An isolated nucleic acid encoding chondroitinase ABC comprising a nucleotide sequence which differs from the nucleotide sequence of SEQ ID NO: 1, due to degeneracy in the genetic code..].

.[.8. An expression vector comprising the nucleic acid as defined in claim 7 operably linked to a regulatory sequence..].

.[.9. A host cell transformed with the expression vector as defined in claim 8..].

.[.10. A method of producing chondroitinase ABC protein comprising: culturing the host cell as in defined in claim 9 under conditions appropriate for expression; and isolating chondroitinase ABC protein from the culture..].

11. An isolated nucleic acid fragment comprising .[.the coding region of chondroitinase ABC and having a nucleotide sequence consisting of.]. nucleotides .[.297-3288.]. .Iadd.73 to 3066 .Iaddend.of SEQ ID NO: 1.

12. An expression vector comprising the nucleic acid of claim 11 operably linked to a regulatory sequence.

13. A host cell transformed with the expression vector of claim 12.

14. A method of producing chondroitinase ABC protein comprising: culturing the host cell as in defined in claim 13 under conditions appropriate for expression; and isolating chondroitinase ABC protein from the culture.

15. An isolated nucleic acid comprising a nucleotide sequence which differs from nucleotides .[.297-3288.]. .Iadd.73 to 3066 .Iaddend.of SEQ ID NO: 1, due to degeneracy in the genetic code.

16. An expression vector comprising the nucleic acid as defined in claim 15 operably linked to a regulatory sequence.

.[.17. An isolated nucleic acid fragment comprising nucleotides 2160-3288 of SEQ ID NO: 1..].

.[.18. An expression vector comprising the nucleic acid as defined in claim 17 operably linked to a regulatory sequence..].

.[.19. An isolated nucleic acid comprising a nucleotide sequence which differs from nucleotides 2160-3288 of SEQ ID NO: 1, due to degeneracy in the genetic code..].

.[.20. An expression vector comprising the nucleic acid as defined in claims operably linked to a regulatory sequence..].

21. An isolated nucleic acid fragment comprising the coding region of the nucleotide sequence of SEQ ID NO: 1.

22. An expression vector comprising the nucleic acid as defined in claim 21 operably linked to a regulatory sequence.

23. A host cell transformed with the expression vector as defined in claim 22.

24. A method of producing chondroitinase ABC protein comprising: culturing the host cell as in defined in claim 23 under conditions appropriate for expression; and isolating chondroitinase ABC protein from the culture.

25. An isolated nucleic acid comprising a nucleotide sequence which differs from the coding region of SEQ ID NO: 1, due to degeneracy in the genetic code.

26. An expression vector comprising the nucleic acid as defined in claim 25 operably linked to a regulatory sequence.

27. A host cell transformed with the expression vector as defined in claim 26.

28. A method of producing chondroitinase ABC protein comprising: culturing the host cell as in defined in claim 27 under conditions appropriate for expression; and isolating chondroitinase ABC protein from the culture.

.Iadd.29. An isolated nucleic acid fragment encoding chondroitinase ABC, wherein the nucleic acid comprises a nucleotide sequence of the insert of pCHS6 obtained from E. coli XL1-Blue/pCHS6 deposited at Accession NO. FERM BP-4170 ..Iaddend.
Description



BACKGROUND OF THE INVENTION

Chondroitin lyase (EC 4.2.2.4) or chondroitinase ABC is an enzyme which catalyzes the depolymerization of chondroitin sulfate. Through .beta.-elimination of 1,4 hexosaminidic bonds, chondroitinase ABC degrades chondroitin, chondroitin 4-sulfate (chondroitin A sulfate), dermatan sulfate (chondroitin B sulfate), chondroitin 6-sulfate (chondroitin C sulfate) and hyaluronate to the respective unsaturated disaccharides (.DELTA.di-OS for chondroitin, .DELTA.di-4S for chondroitin A sulfate, .DELTA.di-4-6S for chondroitin B sulfate and .DELTA.di-6S for chondroitin C sulfate, respectively). The enzyme has been isolated in various strains of bacteria (Neuberg, C. et al., (1914) Biochem. Z. 67: 82-89) (Neuberg, C. et al. (1931) Biochem, Z. 234: 345-346; Yamagata, T. et al., (1968) J. Biol. Chem. 243: 1523-1535) including Proteus vulgaris (Yamagata, T. et al. (1968) J. Biol. Chem. 243: 1523-1535; Thurston, C. F. (1974) J. Gen. Microbiol. 80: 515-522; Sato N. et al. (1986) Agric. Biol. Chem. 50: 1057-1059; Sato N. et al. (1986) Biotechnol. Bioeng. 28: 1707-1712; Sato, N. et al. (1986) J. Ferment. Technol. 64: 155-159).

Chondroitin sulfate consists of alternating .beta. 1-3 glucuronidic and .beta. 1-4 N-acetylgalactosaminidic bonds, and is sulfated at either C-4 or C-6 of the N-acetylgalactosamine pyranose. Chondroitin sulfate is known to be widely distributed in mammalian tissue, such as in skin, cornea, bone and especially in cartilage. Thus, chondroitinase ABC has been used as an experimental reagent for the determination or quantitation of total amount of galactosaminoglycans in the field of orthopedic surgery (Linker, A. et al. (1960) J. Biol. Chem. 235: 3061-3065; Saito, H. et al. (1968) J. Biol. Chem. 243: 1536-1542; Pettipher, E. R. et al. (1989) Arthritis Rheum. 32: 601-607; Caterson, B. et al. (1990) J. Cell Science 97: 411-417; and Seibel, M. J. et al. (1992) Arch. Biochem. Biophys. 296: 410-418).

Recently, chondroitinase ABC has been reported to be a potential reagent for chemonucleolysis, an established treatment for intervertebral disc displacement (Kato, F. et al. (1990) Clin. Orthop. 253: 301-308; Henderson, N. et al. (1991) Spine 16: 203-209). However, for the utilization of chondroitinase ABC as a clinical reagent, there are many problems to be overcome. For example, the preparation of chondroitinase ABC from P. vulgaris requires tedious and intricate procedures, since the cellular content of the enzyme is low. Therefore, an efficient method for the efficient preparation of highly purified chondroitinase ABC is now sought.

SUMMARY OF THE INVENTION

This invention pertains to nucleic acid sequences coding for the chondroitinase ABC gene and isolated chondroitinase ABC protein produced in a host cell transformed with a nucleic acid vector directing the expression of a nucleotide sequence coding for chondroitinase ABC. Chondroitinase ABC prepared by chemical synthesis is also provided. This invention further provides monoclonal and polyclonal antibodies which are specifically reactive with chondroitinase ABC. The isolated chondroitinase ABC can be used in methods of treating intervertebral disc displacement and promoting neurite regeneration or in method of detecting the presence of galactosaminoglycans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-A and 1-B show the primers used for polymerase chain reaction (PCR) amplification of chondroitinase ABC from P. vulgaris genomic DNA.[.;.]..Iadd.. FIG. 1A provides the amino acid sequence of the N-terminal region of purified chondroitinase ABC (SEQ ID NO: 3), the nucleotide sequences of the sense primers (SEQ ID NOS 5 and 6), and the nucleotide sequences of the antisense primers (SEQ ID NOS 7 and 8)..Iaddend.

FIG. 1-B also shows the probe used for plaque hybridization.[.;.]..Iadd.. FIG. 1B provides the amino acid sequence of the N-terminal region of purified chondroitinase ABC (SEQ ID NO: 3), the nucleotide sequence of primer A (SEQ ID NO: 10), the nucleotide sequence of the probe (SEQ ID NO: 9), the nucleotide sequence of primer B (SEQ ID NO: 11), and the full length DNA sequences (SEQ ID NOS 12 and 13)..Iaddend.

FIG. 1-C shows the restriction maps for three recombinant phages and the fragment of phage 11-5 which was subcloned into pSTV29 for sequencing.

FIG. 2 shows the construction of pCHSP, a hybrid plasmid containing the putative promoter region of chondroitinase ABC (SEQ ID NO: 14).

FIG. 3 shows primer extension analysis using a sequencing ladder (SEQ ID NO:15).

FIG. 4 shows the nucleotide sequence of the promoter region of chondroitinase ABC (SEQ ID NO: 16) and the peptide sequence (SEQ ID NO:17).

FIG. 5 shows the construction of plasmids pCHS 6, pCHS.DELTA. 6, and pCHS 26 each of which contains a fragment of the chondroitinase ABC gene.

FIG. 6 shows SDS-PAGE and immunoblot analysis of recombinant chondroitinase ABC protein produced by pCHS.DELTA. 6 transformed E. coli (lane 1); protein produced by pSTV 29 without the chondroitinase ABC gene in E. coli (lane 2); natural chondroitinase ABC produced by P. vulgaris (lane 3); and molecular weight markers (lane 4).

FIG. 7 shows the DNA (SEQ ID NO:1) and amino acid sequence (SEQ ID NO:2) of the chondroitinase ABC gene.[.including non-coding regions.]..

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to nucleic acid sequences coding for chondroitinase ABC, an enzyme which degrades chondroitin A, B, and C. The chondroitinase ABC gene was derived using recombinant DNA techniques. A nucleic acid sequence coding for chondroitinase ABC preferably has the sequence shown in SEQ ID NO: 1 (FIG. 7). The deduced amino acid sequence of chondroitinase ABC is shown in SEQ ID NO:2 (FIG. 7).

Accordingly, one aspect of the invention pertains to an isolated nucleic acid having a nucleotide sequence coding for chondroitinase ABC, fragments thereof, or equivalents thereof. The term nucleic acid as used herein is intended to include such fragments or equivalents. A nucleic acid sequence coding for chondroitinase ABC can obtained from mRNA present in Proteus vulgaris. Nucleic acid sequences coding for chondroitinase ABC can also be obtained from P. vulgaris genomic DNA. The nucleic acid sequence coding for chondroitinase ABC can be obtained using the method disclosed herein or any other suitable technique for isolation and molecular cloning of genes. The nucleic acid sequences of the invention can be DNA or RNA. The preferred nucleic acid is a DNA having the sequence depicted in SEQ ID NO:1 (FIG. 7) or equivalents thereof.

The term equivalent is intended to include nucleotide sequences coding for functionally equivalent chondroitinase ABC proteins. For example, DNA sequence polymorphisms within the nucleotide sequence of chondroitinase ABC (especially those within the third base of a codon) may result in "silent" mutations which do not affect the amino acid sequence of the chondroitinase ABC protein. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequence of chondroitinase ABC will exist. It will be appreciated by one skilled in the art that these variations in one or more nucleotides (up to about 3-4% of the nucleotides) of the nucleic acid sequence coding for chondroitinase ABC may exist due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of the invention. Furthermore, there may be one or more isoforms or related, cross-reacting family members of chondroitinase ABC. Such isoforms or family members are defined as proteins related in function and amino acid sequence to chondroitinase ABC, but encoded by genes at different loci.

A fragment of the nucleic acid sequence coding for chondroitinase ABC is defined as a nucleotide sequence having fewer nucleotides than the nucleotide sequence coding for the entire amino acid sequence of chondroitinase ABC protein. Such fragments encode a catalytically-active fragment of chondroitinase ABC protein which depolymerizes chondroitin A, B, or C. Nucleic acid fragments within the scope of the invention include those capable of hybridizing with nucleic acid from other animal species for use in screening protocols to detect chondroitinase ABC or enzymes that are cross-reactive with chondroitinase ABC. Nucleic acid sequences within the scope of the invention may also contain linker sequences, modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of recombinant chondroitinase ABC or catalytically-active fragments thereof.

This invention also provides expression vectors containing a nucleic acid sequence coding for chondroitinase ABC, operably linked to at least one regulatory sequence. Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence. Regulatory sequences are art-recognized and are selected to direct expression of chondroitinase ABC. Accordingly, the term regulatory sequence includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed.

This invention further pertains to a host cell transformed to express chondroitinase ABC. The host cell may be any prokaryotic or eukaryotic cell. For example, chondroitinase ABC protein may be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO). Other suitable host cells may be found in Goeddel, (1990) supra or one known to those skilled in the art.

Expression in eukaryotic cells such as mammalian, yeast, or insect cells can lead to partial or complete glycosylation and/or formation of relevant inter- or intra-chain disulfide bonds of recombinant protein. Examples of vectors for expression in yeast S. cerivisae include pYepSec1 (Baldari. et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.). Baculovirus vectors available for expression of proteins in cultured insect cells (SF 9 cells) include the pAc series (Smith et al., (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V. A., and Summers, M. D., (1989) Virology 170:31-39). Generally COS cells (Gluzman, Y., (1981) Cell 23:175-182) are used in conjunction with such vectors as pCDM 8 (Aruffo, A. and Seed, B., (1987) Proc. Natl. Acad. Sci. USA 84:8573-8577) for transient amplification/expression in mammalian cells, while CHO (dhfr.sup.- Chinese Hamster Ovary) cells are used with vectors such as pMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195) for stable amplification/expression in mammalian cells. Vector DNA can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, or electroporation. Suitable methods for transforming host cells can be found in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989), and other laboratory textbooks.

Expression in prokaryotes is most often carried out in E. coli with either fusion or non-fusion inducible expression vectors. Fusion vectors usually add a number of NH.sub.2 terminal amino acids to the expressed target gene. These NH.sub.2 terminal amino acids often are referred to as a reporter group. Such reporter groups usually serve two purposes: 1) to increase the solubility of the target recombinant protein; and 2) to aid in the purification of the target recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the reporter group and the target recombinant protein to enable separation of the target recombinant protein from the reporter group subsequent to purification of the fusion protein. Such enzymes include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-tranferase, maltose E binding protein, or protein A, respectively, to the target recombinant protein.

Inducible non-fusion expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). While target gene expression relies on host RNA polymerase transcription from the hybrid trp-lac fusion promoter in pTrc, expression of target genes inserted into pET 11d relies on transcription from the T7 gn10-lac 0 fusion promoter mediated by coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident g prophage harboring a T7 gn1 under the transcriptional control of the lacUV 5 promoter.

One strategy to maximize recombinant chondroitinase ABC expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Another strategy would be to alter the nucleic acid sequence of the chondroitinase ABC gene to be inserted into an expression vector so that the individual codons for each amino acid would be those preferentially utilized in highly expressed E. coli proteins (Wada et al., (1992) Nuc. Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

The nucleic acid sequences of the invention can also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071, incorporated by reference herein).

This invention further pertains to methods of producing chondroitinase ABC protein. For example, a host cell transformed with a nucleic acid vector directing expression of a nucleotide sequence coding for chondroitinase ABC protein can be cultured under appropriate conditions to allow expression of chondroitinase ABC to occur. The protein may be secreted and isolated from a mixture of cells and medium containing chondroitinase ABC protein. Alternatively, the protein may be retained cytoplasmically and the cells harvested, lysed and the protein isolated. The culture includes host cells, media and other byproducts. Suitable mediums for cell culture are well known in the art. Chondroitinase ABC protein can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for chondroitinase ABC or fragment thereof.

Another aspect of the invention pertains to isolated chondroitinase ABC protein. The term "chondroitinase ABC" or "chondroitinase ABC protein" is intended to include functional equivalents thereof and catalytically-active fragments thereof. The term functional equivalent is intended to include proteins which differ in amino acid sequence from the chondroitinase ABC sequence depicted in SEQ ID NO:2 (FIG. 7) but where such differences result in a modified protein which functions in the same or similar manner as chondroitinase ABC or which has the same or similar characteristics of chondroitinase ABC. For example, a functional equivalent of chondroitinase ABC may have a modification such as a substitution, addition or deletion of an amino acid residue which is not directly involved in the enzyme activity of chondroitinase ABC (i.e., the ability of chondroitinase ABC to depolymerize chondroitin 4-sulphate, chondroitin 6-sulfate, and dermatan sulfate). Various modifications of the chondroitinase ABC protein to produce functional equivalents of chondroitinase ABC are described in detail herein.

The term isolated as used herein refers to chondroitinase ABC protein substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Such chondroitinase ABC protein is also characterized as being essentially free of all other P. vulgaris proteins. Accordingly, an isolated chondroitinase ABC protein is produced recombinantly or synthetically and is substantially free of cellular material and culture medium or substantially free of chemical precursors or other chemicals and is essentially free of all other P. vulgaris proteins.

Fragments of chondroitinase ABC which depolymerize chondroitin A, B, or C (referred to herein as catalytically-active fragments) may be obtained, for example, by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid sequence of chondroitinase ABC coding for such peptides. In addition, fragments can be chemically synthesized using techniques known in the art such as by conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, the chondroitinase ABC protein may be arbitrarily divided into fragments of desired length. The fragments can be produced (recombinantly or by chemical synthesis) and tested to determine their enzymatic activity, for example, by contacting the fragment with chondroitin A, B, or C under conditions which allow for depolymerization and determining the extent to which depolymerization occurs.

It is possible to modify the structure of the chondroitinase ABC protein for such purposes as increasing solubility, enhancing therapeutic efficacy, or stability (e.g., shelf life ex vivo and resistance to proteolytic degradation in vivo). Such modified proteins or analogues are considered functional equivalents of the chondroitinase ABC protein as defined herein.

To facilitate purification and potentially increase solubility of the chondroitinase ABC protein, it is possible to add an amino acid reporter group to the protein backbone. For example, hexa-histidine can be added to the protein for purification by immobilized metal ion affinity chromatography (Hochuli, E. et al., (1988) Bio/Technology 6:1321-1325). In addition, to facilitate isolation of chondroitinase ABC protein free of irrelevant sequences, specific endoprotease cleavage sites can be introduced between the sequences of the reporter group and the protein or peptide.

Another aspect of the invention pertains to an antibody specifically reactive with chondroitinase ABC. The antibodies of this invention can be used to isolate the naturally-occurring or native form of chondroitinase ABC or to neutralize the enzyme so that it is unable to depolymerize chondroitin. For example, by using isolated chondroitinase ABC protein based on the cDNA sequence of chondroitinase ABC, anti-protein/anti-peptide antisera or monoclonal antibodies can be made using standard methods. A mammal such as a mouse, a hamster or a rabbit can be immunized with an immunogenic form of the isolated chondroitinase ABC protein (e.g., chondroitinase ABC protein or an antigenic fragment which is capable of eliciting an antibody response). Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. The chondroitinase ABC protein or fragment thereof can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum standard ELISA or other immunoassay can be used with the immunogen as antigen to assess the levels of antibodies.

Following immunization, anti-chondroitinase ABC antisera can be obtained and, if desired, polyclonal anti-chondroitinase ABC antibodies isolated from the serum. To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the art, for example the hybridoma technique originally developed by Kohler and Milstein, Nature (1975) 256:495-497, as well as other techniques such as the human B-cell hybridoma technique (Kozbar et al., Immunology Today (1983) 4:72) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy (1985) Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the chondroitinase ABC protein and the monoclonal antibodies isolated.

The term antibody as used herein is intended to include fragments thereof which are also specifically reactive with the chondroitinase ABC protein or fragment thereof. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab').sub.2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab').sub.2 fragment can be treated with papain to reduce disulfide bridges to produce Fab' fragments. The antibody of the present invention is further intended to include bispecific and chimeric molecules having an anti-chondroitinase ABC portion.

This invention provides therapeutic compositions for the treatment of intervertebral displacement or nerve damage. The composition comprises a therapeutically active amount of chondroitinase ABC protein and a pharmaceutically acceptable carrier. Administration of the therapeutic compositions of the present invention to an individual to be treated can be carried out using known procedures, at dosages and for periods of time effective to depolymerize chondroitin A, B, or C. A therapeutically active amount of chondroitinase ABC protein may vary according to factors such as the amount of chondroitin to be eliminated, the age, sex, and weight of the individual, and the ability of the chondroitinase ABC protein to depolymerize the chondroitin. Dosage regima may be adjusted to provide the optimum therapeutic response.

The active compound (i.e., chondroitinase ABC protein) may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.). If the active compound is administered by injection, for example, about 100 units of active compound (i.e., chondroitinase ABC protein) per dosage unit may be administered to treat intervertebral disc displacement. One unit is the amount of enzyme needed to mediate the release of one micromole of 4,5 unsaturated disaccharide from a substrate of chondroitin C sulfate per minute at 37.degree. C., pH 6.0.

The active compound may be administered parenterally. Dispersions can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating active compound (i.e., chondroitinase ABC protein) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient (i.e., protein) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

As used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic composition is contemplated. Supplementary active compounds can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the elimination of chondroitin A, B, or C.

Isolated chondroitinase ABC protein (i.e., chondroitinase ABC produced recombinantly or by chemical synthesis) is essentially free of all other P. vulgaris proteins. Such protein is of a consistent, well-defined composition and biological activity for use in preparations which can be administered for therapeutic purposes (e.g., to treat intervertebral disc displacement). Such proteins can also be used as diagnostic reagents or in the study of the mechanism of chondroitinase ABC and to design modified derivatives or analogs useful in the depolymerization of chondroitin.

This invention also provides a method of treating intervertebral disc displacement by chemonucleolysis using isolated chondroitinase ABC. Chondroitinase ABC is a particularly useful enzyme for the selective chemonucleolysis of the nucleus pulposus (See, for example, U.S. Pat. No. 4,696,816). The nucleus pulposus is made up of proteoglycans and collagen fibers. Chondroitinase ABC attacks the polysaccharide side chains of the proteoglycans and reduces the swelling of the disc without affecting the structural collagen components or degrading the protein element of the proteoglycan. The disc then shrinks and pressure on the spinal cord is relieved. Thus, to treat intervertebral disc displacement, an active amount of the chondroitinase ABC protein of the invention can be applied to the affected area. For example, 100 units of isolated chondroitinase ABC can be injected into the center of a disc by the standard technique of intradiscal injection (Brown, Intradiscal Therapy, Year Book Medical Publishers, Inc., Chicago, 1983).

The invention further provides a method of treating nerve damage by applying an active amount of the chondroitinase ABC protein of the invention to the affected area to degrade chondroitin-6-sulfate proteoglycans. It has been found that chondroitin 6-sulfate proteoglycans inhibit regeneration of neurites in the adult vertebrate central nervous system (McKeon et al., J. Neurosci 11:3398-3411 (1991)). By removing chondroitin 6-sulfate proteoglycans from the point of injury, it is possible to promote neurite regeneration. For example, a therapeutically effective amount of isolated chondroitinase ABC can be applied to the point of injury in an individual to degrade inhibitory chondroitin 6-sulfate proteoglycans. More than one dose may be administered as indicated by the exigencies of the therapeutic situation.

The chondroitinase ABC protein of the invention can also be used as a diagnostic reagent for detecting the presence of a galactosaminoglycan, such as chondroitin sulfate. For example, the chondroitinase ABC protein can be used as a reagent for determining or quantitating the amount of galactosaminoglycan in a mammalian tissue, such as skin, cornea, bone or cartilage (See e.g., Linker, A. et al. (1960) J. Biol. Chem. 235: 3061-3065; Saito, H. et al. (1968) J. Biol. Chem. 243: 1536-1542; Pettipher, E. R. et al. (1989) Arthritis Rheum. 32: 601-607; Caterson, B. et al. (1990) J. Cell Science 97: 411-417; and Seibel, M. J. et al. (1992) Arch. Biochem. Biophys. 296: 410-418). To determine the presence of chondroitin sulfate in a mammalian tissue, chondroitinase ABC protein can be contacted with a sample of the tissue and the presence or amount of chondroitin sulfate determined using methods well known in the art.

The invention is further illustrated by the following examples which should not be construed as further limiting the subject invention. The contents of all references and published patent applications cited throughout this application are hereby incorporated by reference. The following methods and materials were used throughout the examples discussed below.

Materials and Methods

Bacterial strains, plasmid and phage P. vulgaris IFO3988 was provided by the Institute for Fermentation, Osaka, Japan. E. coli P2392 (hsdR514(r.sup.k-, m.sup.k+), supE44, supF58, lacY1 or (lacIZY), galT22, metB1, trpR55, (P2)) was used as the lysogen for P2 phage. EMBL3 vector was purchased from Toyobo Co., Ltd., Japan. PCR products were ligated with pT7 Blue T-vector(Takara Shuzo Co., Ltd., Japan). E. coli JM109(recA1, endA1, gyrA96, thi, hsdR17(r.sup.k-, m.sup.k+), supE44, relA1, .lamda.-, .DELTA.(lac-proB), (F', proAB, lacIq M15, traD36) was used as the host strain for pMC1871 promoter selection vector (Pharmacia LKB, Japan). E. coli XL1-Blue(endA1, hsdR17(r.sup.k-, m.sup.k+), supE44, thi-1, recA1, gyrA96, relA1, A(lac), (F', proAB, lac, (lacZ.DELTA.M15, Tn10(tetr)) (Int'l Dep. No. FERM BP-4170). E. coli XL1-Blue is a host cell for both pSTV28, and pSTV29 (Takara Shuzo Co., Ltd., Japan).

N-terminal amino acid sequence Chondroitinase ABC was purified as described previously (Sato, N. et al. (1986) Agric. Biol. Chem. 50: 1057-1059). The N-terminal amino acid sequence of chondroitinase ABC was sequenced by automatic Edman degradation on a gas-phase sequencer (Applied Biosystem, Foster, Calif.). The sequence of the N-terminal region of chondroitinase ABC was Ala-Thr-Ser-Asn-Pro-Ala-Phe-Asp-Pro-Lys-Asn-Leu-Met-Gln-Ser-Glu-Ile-Tyr (18 amino acid residues) (SEQ ID NO:3) The double stranded DNA sequence is shown in FIG. 1-B (SEQ ID NOS:12-13).

Isolation of DNA and synthesis of nucleic acid, primer, and probe Isolation of chromosomal DNA of P. vulgaris was carried out by the standard method (Silhavy, T. J. et al. (1984) Experiments with Gene Fusion, Cold Spring Harbor Laboratory Press). Oligonucleotides used as primers and probe were synthesized with the DNA synthesizer, Cyclone Plus (Milligene/Biosearch, Bedford, Mass.).

Construction and screening of the gene library SauIII AI-partially digested fragments of total DNA were ligated to the BamHI site in .lamda.EMBL3 arms according to Frischauf et al. (J. Mol. Biol. 170: 827-842 (1983)). The ligation mixture was packaged in vitro and transfected to E. coli P2392 according to the instructions of the suppliers (Stratagene, La Jolla, Calif.).

PCR amplification Primers for the chondroitinase ABC gene were designed according to the amino acid sequence of the chondroitinase ABC N-terminal region (SEQ ID NO:3)(FIG. 1-A). The primers were as follows 5'-GCNACNUCNAAYCCNGC-3' (P-1, sense)(SEQ ID NO:5); 5'-GCNACNAGYAAYCCNGC-3' (P-2, sense)(SEQ ID NO:6); 5'-UACGUYAGNCUYUADAU-3' (P-3, antisense)(SEQ ID NO:7); 5'-UACGUYUCRCUYUADAU-3' (P-4 antisense)(SEQ ID NO:8) (FIG. 1-A).

PCR was performed using a GeneAmp Kit (Takara Shuzo Co., Ltd., Japan) in a final volume of 100 .mu.l which contained: 1 .mu.g of genomic DNA solution, 10 .mu.l of 10.times. PCR reaction buffer, 16 .mu.l of 1.25 mM dNTP mixture, 0.6 nmol of mixed primers and 2.5 units of Taq DNA polymerase (Takara Shuzo Co., Ltd., Japan). The mixture was subjected to PCR amplification using the DNA thermal cycler (GeneAmp PCR System 9600, Perkin-Elmer/Cetus, Norwalk, Conn.) for 28 cycles. Each cycle was 1 minute at 93.degree. C.(denaturation), 1.5 minutes at 50.degree. C.(annealing) and 0.5 minute at 72.degree. C.(elongation). PCR products were analyzed by electrophoresis through a 5% agarose gel (Nusieve GTG agarose, FMC Bioproducts, Rockford, Me.) and the 54 bp fragment encoding 17 amino acids of N-terminal region was cut out of the gel. Gel-purified PCR products were directly cloned into pT7 Blue PCR vector.

DNA Sequencing and Isolation of the Chondroitinase ABC Gene

Double-stranded plasmids purified by polyethylene glycol were denatured with alkali and sequenced by dideoxynucleotide chain termination method (Sanger, R. et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463-5467) using the sequence system, Hitachi WS10A Personal Sequencer (Hitachi Electronics Co., Ltd., Japan) Direct sequencing was done according to the method of Gyllesten & Erlich (Gyllensten, U. (1989) in PCR Technology, Erlich, H. A., Ed., Stockton Press, New York, pp. 45-60). PCR screening was carried out by the method of Olson et al.(Science 245: 1434-1435 (1989)). Plaque hybridization (Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press) and Southern hybridization (Southern, E. M. (1975) J. Mol. Biol. 8: 503-517) were performed as outlined in the instructions of the supplier(Amersham Japan).

Primer extension analysis A 21-mer oligonucleotide(5'-CTA ATG GGT TAT TTT GTG CAA-3') (SEQ ID NO:4) complementary to the 5'-end (nucleotides 355-375) of the chondroitinase ABC gene was used as a primer. It was labeled with .gamma.-.sup.32P ATP (Amersham Japan) using polynucleotide kinase (Toyobo Co., Ltd., Japan). Total RNA of P. vulgaris was prepared according to the method of Aiba (J. Biol. Chem. 260: 3063-3070 (1985)). The labeled primer and 5 .mu.g of total RNA were coprecipitated with ethanol. After annealing at 25.degree. C. for 6 hours in a hybridization buffer (80% formamide, 40 mM PIPES(pH 6.4), 1 mM EDTA and 400 mM NaCl), 250 mM NaCl, 50 mM sodium acetate(pH 4.6), 4.5 mM ZnSO.sub.4, 100 .mu.g/ml heat-denatured salmon testes DNA and 15 unit/.mu.l reverse transcriptase of Rous associated virus 2 (Takara Shuzo Co., Ltd., Japan) were added to the mixture. The primer extension reaction was carried out at 37.degree. C. for 60 minutes.

Culture conditions Cells of E. coli XL1-Blue carrying recombinant plasmid were grown in 3 ml of LB broth(1% tryptone, 0.5% yeast extract, 1% NaCl, 25 .mu.g/ml of chloramphenicol (pH 7.5)) at 37.degree. C. for 16 hr with reciprocation (120 rpm, 5 cm stroke). The cells were harvested by centrifugation and washed twice with 0.85% saline solution. Cells were transferred to 100 ml of chondroitin 6-sulfate (Taiyo Fishery Co., Ltd., Japan) medium(0.7% K.sub.2HPO.sub.4, 0.3% KH.sub.2PO.sub.4, 0.01% MgSO.sub.4.7H.sub.2O, 0.1% (NH.sub.4).sub.2SO.sub.4, 0.1% yeast extract, 0.3% chondroitin 6-sulfate, 0.01% glucose, 25 .mu.g/ml chloramphenicol (pH 7.5)) or glucose medium (composition is the same as that of chondroitin medium except that glucose (0.3%) was used as a carbon source) to make a final concentration of A.sub.610=0.05. After incubation for 3 days at 37.degree. C. with reciprocation, the cells were removed by centrifugation and degradation products of chondroitin 6-sulfate in the culture fluid were determined. The cells harvested from chondroitin and glucose media were washed twice with 50 mM Tris-HCl buffer (pH 8.0) and sonicated at 90 kHz for 5 minutes at 0.degree. C. The cell debris were removed by centrifugation at 20,000 g for 30 minutes, and the supernatant was used for the assay of chondroitinase ABC.

Enzyme assay Chondroitinase ABC was assayed as described previously (Sato, N. et al. (1986) J. Ferment. Technol. 64: 155-159). The assay mixture (3 ml) containing 0.5% chondroitin 6-sulfate, 100 mM potassium phosphate buffer (pH 8.0) and cell extract, was incubated at 37.degree. C. for 10 minutes, and the amount of N-acetylgalactosamine end group formed was determined by the method of Reissig (J. Biol. Chem. 217: 959-966 (1955)). Activity was expressed as the quantity of enzyme that catalyzed the formation of 1 .mu.mol of unsaturated disaccharide (.DELTA.di-6S) from chondroitin 6-sulfate per minute at 37.degree. C.

Western blot analysis IgG specific to chondroitinase ABC was isolated from antisera raised in guinea pig using the technique described previously (Sato, N. et al. (1988) Biotechnol. Appl. Biochem. 10: 385-393). Proteins in crude cell extracts prepared from E. coli transformant were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (Sato, N. et al. (1986) Agric. Biol. Chem. 50: 1057-1059). Western blotting procedures were described previously (Sato, N. et al. (1989) Appl. Microbiol. Biotechnol. 30: 153-159).

EXAMPLE 1

Isolation and Sequence Determination of the Chondroitinase ABC Gene

According to the amino acid sequence of the N-terminal region of purified chondroitinase ABC (Ala-Thr-Ser-Asn-Pro-Ala-Phe-Asp-Pro-Lys-Asn-Leu-Met-Gln-Ser-Glu-Ile-Tyr (FIG. 1-A)(SEQ ID NO:3)), a set of degenerate oligo mixed primers (5'-GCNACNUCNAAYCCNGC-3' (P-1, sense)(SEQ ID NO:5); 5'-GCNACNAGYAAYCCNGC-3' (P-2, sense)(SEQ ID NO:6); 5'-UACGUYAGNCUYUADAU-3' (P-3, antisense)(SEQ ID NO:7); 5'-UACGUYUCRCUYUADAU-3' (P-4 antisense)(SEQ ID NO:8))(FIG. 1-A) were synthesized as follows. To determine the appropriate primers for sequencing, PCR amplification of a combination of primers P-1(SEQ ID NO:5), P-2(SEQ ID NO:6) (sense) and P-3(SEQ ID NO:7), P-4(SEQ ID NO:8) (antisense) was performed. After agarose gel electrophoresis of these PCR products, a 54 bp fragment was extracted and directly inserted into pT7 Blue PCR vector, and the inserted fragment was sequenced. The nucleotide sequence of this fragment was found to be identical to the N-terminal amino acid sequence (FIG. 1-B) (SEQ ID NO:3). Then, using primer A (5'-GCAACCAGCAATCCTGCA-3')(SEQ ID NO:10), primer B (.[.5.]. .Iadd.3.Iaddend.'-GACTACGTCAGGCTTT.[.T.]. .Iadd.A.Iaddend.AAT-.[.3.]. .Iadd.5.Iaddend.')(SEQ ID NO:11) (FIG. 1-B) and 1 .mu.g of P. vulgaris genomic DNA as a template, PCR analysis was performed and PCR products were analyzed by agarose gel electrophoresis. No non-specific PCR products were observed.

We then diluted .gamma.EMBL3 recombinant phage stock library. The diluted library was used for PCR screening. An unique 54 bp fragment was clearly detected until the dilution of 1/10.sup.3(2.times.10.sup.5 pfu) phage stock solution as a template. The diluted phage solution was divided by 1/10(2.times.10.sup.4 pfu) and was infected into E. coli P2392. They were then subjected to plaque hybridization using .sup.32P-labeled probe (5'-CATTTGATCCTAAAAATCTGATGCA-3')(SEQ ID NO:9)(FIG. 1-B). The recombinant phages were chosen at random and analyzed by restriction mapping and Southern blotting. All phages contained common 4.2 kb EcoRV-EcoRI, 1.1 kb ClaI, and 2.0 kb EcoRV-HindIII fragments which hybridized strongly with the probe(SEQ ID NO:9). The restriction maps of three types of SalI fragments are shown in FIG. 1-C. Southern hybridization patterns of restricted genomic DNA from P. vulgaris matched the restriction map of these fragments. This result suggests that the 4.2 kb EcoRV-EcoRI fragment originated in the P. vulgaris genome, and therefore, the chondroitinase ABC gene exists as a single copy. When purified chondroitinase ABC from P. vulgaris was analyzed by SDS-PAGE, two types of chondroitinase ABC protein, one 100 kd protein and one subunit-like protein at 80 kd and 20 kd, were observed. The amino acid composition of the 100 kd protein and the subunit-like protein (80 kd and 20 kd) were quite similar, and the N-terminal amino acid sequences of the 100 kd and 20 kd proteins were identical. The results indicate that the two forms of chondroitinase ABC were not derived from two separate chondroitinase ABC genes.

The 5.2 kb SalI-EcoRI fragment in the recombinant .gamma.EMBL3 (No. 11-5) (FIG. 1-C) was subcloned into pSTV29 for sequencing and the resulting hybrid plasmid was designated pCHS6. The entire 3,063 bp nucleotide sequence of the coding region for the chondroitinase ABC gene as well as 224 and 200 nucleotides of the upstream and downstream regions, respectively, and the deduced amino acid sequence of chondroitinase ABC are shown in FIG. 7 (SEQ ID NO:1). The 25-mer oligonucleotide probe (SEQ ID NO:9) hybridized to nucleotide 314-337. The 16/18 nucleotide of primer A and the 17/18 nucleotide of primer B were the same in nucleotides 297-313 and 333-349. The G+C content of the chondroitinase ABC gene was 38.6%. The open reading frame encoded a polypeptide with a molecular weight of 115,218, which represents a precursor polypeptide containing a signal peptide sequence that is subsequently cleaved off at Ala.sup.24-Ala.sup.25 during secretion of the mature chondroitinase ABC protein having a molecular weight of 112,365.

EXAMPLE 2

Analysis of the transcription region of the chondroitinase ABC gene In order to confirm the potential promoter region of the chondroitinase ABC gene, we amplified the region of nucleotide 112-283 using PCR. The PCR product was blunt-ended with T4 DNA polymerase and inserted into the SmaI site of the promoter selection vector, pMC 1871, and the hybrid plasmid, designated pCHSP, was introduced into E. coli JM109 (FIG. 2)(SEQ ID NO:14). The transformant was then cultured in an LB medium containing 25 .mu.g/ml tetracycline at 37.degree. C. for 14 hr, and .beta.-galactosidase activity was assayed (Table I). Although the .beta.-galactosidase activity of the E. coli transformant carrying pMC1871 was not detectable, the E. coli transformant carrying pCHSP produced .beta.-galactosidase. This result indicates that the chondroitinase ABC gene can function as a promoter in E. coli cells. However, there is a possibility that the promoter recognized in E. coli cells may not be the promoter in P. vulgaris. To confirm that the promoter is recognized in P. vulgaris, primer extension analysis was carried out (FIG. 3) (SEQ ID NO:15). The transcription start point was localized to an adenosine 41 bp upstream from the start codon, ATG (FIG. 4) (SEQ ID NO:16). The potential pribnow box (TTTAAT) (nucleotides 169-174) was located 12 bp upstream from the transcription start point (FIG. 4) (SEQ ID NO: 16). However, the -35 consensus sequence was not found near 35 bp upstream of the start point except for 47 bp upstream of the start point (TAGGCA) (FIG. 4) (SEQ ID NO:16). The Shine-Dalgarno ribosomal binding site (AGGAGA) (nucleotides 213-218) was found 9 bp upstream from the initiation codon, ATG (FIG. 4) (SEQ ID NO: 16). A terminator-like palindrome sequence consisting of an 11 nucleotide stem with a 4 nucleotide loop structure (stacking energy 24 kcal/mol) was located 9 nucleotides downstream from the stop codon, TGA (FIG. 4) (SEQ ID NO:16). Judging from the secondary structure prediction, this stem-loop structure resembles a .sigma.-dependent transcription terminator.

TABLE-US-00001 TABLE I .beta.-Galactosidase productivity of E-coli transformants .beta.-Galactosidase activity Strain Activity Specific activity/ E. coli JM109 (U/mi-culture) (U/mg-protein) /pMC1871 0 0 /pCHSP 0.2 0.4 1 U is defined as the amount that produced 1 .mu.mol of .alpha.-nitrophenol per h.

EXAMPLE 3

Production of chondroitinase ABC from E. coli transformant To demonstrate that the isolated gene codes for chondroitinase ABC, we constructed pCHS.DELTA.6 and pCHS26 (FIG. 5). pCHS.DELTA.6 was constructed by removing the SalI-EcoRV region (about 1 kb) upstream from the promoter region from the chondroitinase ABC gene. While pCHS26 was constructed by removing the HindIII-EcoRI region which corresponded to about one third of the 3'-terminal region of the chondroitinase ABC structural gene. These plasmids (pCHS6, pCHS.DELTA.6 and pCHS26) were introduced into E. coli XL 1-Blue, and E. coli transformants were cultured in chondroitin or glucose medium, and chondroitinase ABC activities were assayed using the crude extract. The culture fluids of the chondroitin medium were also analyzed to determine degradation products of chondroitin 6-sulfate (Table II). The E. coli transformant carrying pCHS6 (containing a 1.0 kb fragment upstream from the promoter) produced the chondroitinase ABC when cultured in chondroitin medium, however, no chondroitinase ABC activity was observed when the transformant was cultured in glucose medium. In contrast, the E. coli transformant carrying pCHS.DELTA.6 produced chondroitinase ABC when cultured in either chondroitin or glucose media. The production levels of chondroitinase ABC, cultured in chondroitin media, were 2.6 fold(/pCHS6) and 187 fold(/pCHS.DELTA.6) higher than that of P. vulgaris. Even cultured in glucose medium, the production level of chondroitinase ABC in the E. coli transformant carrying pCHS.DELTA.6 was 187 fold higher than that of P. vulgaris cultured in chondroitin medium. This result suggests that the regulatory sequence might be in the SalI-EcoRV region. Although chondroitin 6-sulfate added to the medium was degraded (p/CHS6 and /pCHS.DELTA.6), E. coli transformants were not able to utilize chondroitin sulfate as a carbon source.

TABLE-US-00002 TABLE II Chondroitinase ABC Activity of E. coli Transformants Intracellular chondroitinase ABC activity Chondroitin medium Glucose Cultured medium (0.3%) medium (0.3%) Amount of 4,5.DELTA. Specific.sup.b Specific chondroitin-6 Strain Activity.sup.a activity Actiity activity (.mu.g/ml-culture) E. coli 0 0 0 0 0 XL1-Blue /pSTV29 0 0 0 0 0 /pCHS6 4.1 .times. 10.sup.-3 1.6 .times. 10.sup.-2 0 0 192.7 /pCHS26 0 0 0 0 0 /pCHS.DELTA.6 0.3 1.2 0.3 0.5 1542.4 P. vulgaris 1.6 .times. 10.sup.-3 1.2 .times. 10.sup.-2 0 0 1738.4 .sup.a1 U: enzyme activity producing 1 .mu.mol, 4,5.DELTA. chondroitin-6 per min .sup.bU/mg-protein

It has been reported that the Bacteriodes thetaiotaomicron chondroitin lyase II gene is adjacent to the chondrosulfatase gene which may be a part of an operon (Guthrie, E. P. et al. (1987) J. Bacteriol. 169: 1192-1199). These same investigators reported that the promoter for this gene recognized in E. coli may not be the promoter from which the chondroitin lyase II gene is transcribed from in B. thetaiotaomicron (Ld.) In fact, a putative open reading frame 12 bp upstream from the initiation codon, ATG, was found in the chondroitinase ABC gene (FIG. 4) (SEQ ID NO: 16). However, primer extension analysis revealed that the transcription start point is located 41 bp upstream from the initiation codon in P. vulgaris(FIG. 3) (SEQ ID NO: 15). Even though the chondroitinase ABC gene from P. vulgaris cells was also part of an operon, chondroitinase ABC gene was transcribed 41 bp upstream from the initiation codon in P. vulgaris cells.

The secondary structure of chondroitinase ABC was estimated by the method of Chou and Fasman (Annu. Rev. Biochem. 47: 251-276 (1978)). A highly complex region was found between amino acid residues 450 and 850. The pCHS26 lacks one-third of the chondroitinase ABC gene encoding the C-terminal region (amino acid residues 646-1021). Removing this region of the enzyme caused the disappearance of chondroitinase ABC activity (Table II). This result suggests that there might be an active site in this region.

Recombinant chondroitinase ABC produced by E. coli carrying pCHS.DELTA.6 was analyzed by SDS-PAGE followed by immunoblotting (FIG. 6). The immunoblotting patterns of recombinant and native chondroitinase ABC (100 kd) were quite similar. Our previous report showed chondroitinase ABC purified from P. vulgaris was a subunit structure consisting of a 90 kd and a 20 kd protein by SDS-PAGE (Sato, N. et al. (1986) Agric. Biol. Chem. 50: 1057-1059), because this subunit protein would not be separated even using gel filtration and other chromatographic techniques. However, by analysis of the N-terminal sequence, we found that the 100 kd protein and the 20 kd protein had the same N-terminal amino acid sequence. By immunoblot analysis, the 80 kd protein also reacts with IgG specific to the 100 kd protein. Furthermore, genomic restriction analysis suggested that chondroitinase ABC gene was a single gene. When we extracted the 100 kd band of chondroitinase ABC from the acrylamide gel and electrophoresed it again in SDS-PAGE, 80 kd and 20 kd bands appeared. The purified chondroitinase ABC contained no protease activity. These results suggest that chondroitinase ABC was partially digested not enzymatically, but physically in the course of sample preparation for SDS-PAGE.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

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DNAProteus vulgarisCDS(63) g ata ttt cgt ttt act gca ctt gca atg aca ttg ggg cta tta 48Met Pro Ile Phe Arg Phe Thr Ala Leu Ala Met Thr Leu Gly Leu Leucg cct tat aac gcg atg gca gcc acc agc aat cct gca ttt gat 96Ser Ala Pro Tyr Asn Ala Met Ala Ala Thr Ser Asn Pro Ala Phe Asp 2cct aaa aat ctg atg cag tca gaa att tac cat ttt gca caa aat aac Lys Asn Leu Met Gln Ser Glu Ile Tyr His Phe Ala Gln Asn Asn 35 4 tta gca gac ttc tca tca gat aaa aac tca ata cta acg tta tct Leu Ala Asp Phe Ser Ser Asp Lys Asn Ser Ile Leu Thr Leu Ser 5gat aaa cgt agc att atg gga aac caa tct ctt tta tgg aaa tgg aaa 24s Arg Ser Ile Met Gly Asn Gln Ser Leu Leu Trp Lys Trp Lys65 7ggt ggt agt agc ttt act tta cat aaa aaa ctg att gtc ccc acc gat 288Gly Gly Ser Ser Phe Thr Leu His Lys Lys Leu Ile Val Pro Thr Asp 85 9 gaa gca tct aaa gca tgg gga cgc tca tct acc ccc gtt ttc tca 336Lys Glu Ala Ser Lys Ala Trp Gly Arg Ser Ser Thr Pro Val Phe Ser tgg ctt tac aat gaa aaa ccg att gat ggt tat ctt act atc gat 384Phe Trp Leu Tyr Asn Glu Lys Pro Ile Asp Gly Tyr Leu Thr Ile Asp gga gaa aaa ctc att tca acc agt gag gct cag gca ggc ttt aaa 432Phe Gly Glu Lys Leu Ile Ser Thr Ser Glu Ala Gln Ala Gly Phe Lys aaa tta gat ttc act ggc tgg cgt gct gtg gga gtc tct tta aat 48s Leu Asp Phe Thr Gly Trp Arg Ala Val Gly Val Ser Leu Asn aac gat ctt gaa aat cga gag atg acc tta aat gca acc aat acc tcc 528Asn Asp Leu Glu Asn Arg Glu Met Thr Leu Asn Ala Thr Asn Thr Ser gat ggt act caa gac agc att ggg cgt tct tta ggt gct aaa gtc 576Ser Asp Gly Thr Gln Asp Ser Ile Gly Arg Ser Leu Gly Ala Lys Val agt att cgt ttt aaa gcg cct tct aat gtg agt cag ggt gaa atc 624Asp Ser Ile Arg Phe Lys Ala Pro Ser Asn Val Ser Gln Gly Glu Ile 2tc gac cgt att atg ttt tct gtc gat gat gct cgc tac caa tgg 672Tyr Ile Asp Arg Ile Met Phe Ser Val Asp Asp Ala Arg Tyr Gln Trp 222t tat caa gta aaa act cgc tta tca gaa cct gaa att caa ttt 72p Tyr Gln Val Lys Thr Arg Leu Ser Glu Pro Glu Ile Gln Phe225 234c gta aag cca caa cta cct gta aca cct gaa aat tta gcg gcc 768His Asn Val Lys Pro Gln Leu Pro Val Thr Pro Glu Asn Leu Ala Ala 245 25t gat ctt att cgc caa cgt cta att aat gaa ttt gtc gga ggt gaa 8sp Leu Ile Arg Gln Arg Leu Ile Asn Glu Phe Val Gly Gly Glu 267g aca aac ctc gca tta gaa gag aat atc agc aaa tta aaa agt 864Lys Glu Thr Asn Leu Ala Leu Glu Glu Asn Ile Ser Lys Leu Lys Ser 275 28t ttc gat gct ctt aat att cac act tta gca aat ggt gga acg caa 9he Asp Ala Leu Asn Ile His Thr Leu Ala Asn Gly Gly Thr Gln 29ga cat ctg atc act gat aaa caa atc att att tat caa cca gag 96g His Leu Ile Thr Asp Lys Gln Ile Ile Ile Tyr Gln Pro Glu33at ctt aac tcc caa gat aaa caa cta ttt gat aat tat gtt att tta Leu Asn Ser Gln Asp Lys Gln Leu Phe Asp Asn Tyr Val Ile Leu 325 33t aat tac acg aca tta atg ttt aat att agc cgt gct tat gtg ctg Asn Tyr Thr Thr Leu Met Phe Asn Ile Ser Arg Ala Tyr Val Leu 345a gat ccc aca caa aag gcg caa cta aag cag atg tac tta tta Lys Asp Pro Thr Gln Lys Ala Gln Leu Lys Gln Met Tyr Leu Leu 355 36g aca aag cat tta tta gat caa ggc ttt gtt aaa ggg agt gct tta Thr Lys His Leu Leu Asp Gln Gly Phe Val Lys Gly Ser Ala Leu 378a acc cat cac tgg gga tac agt tct cgt tgg tgg tat att tcc Thr Thr His His Trp Gly Tyr Ser Ser Arg Trp Trp Tyr Ile Ser385 39ta tta atg tct gat gca cta aaa gaa gcg aac cta caa act caa Leu Leu Met Ser Asp Ala Leu Lys Glu Ala Asn Leu Gln Thr Gln 44at gat tca tta ctg tgg tat tca cgt gag ttt aaa agt agt ttt Tyr Asp Ser Leu Leu Trp Tyr Ser Arg Glu Phe Lys Ser Ser Phe 423g aaa gta agt gct gat agc tct gat cta gat tat ttc aat acc Met Lys Val Ser Ala Asp Ser Ser Asp Leu Asp Tyr Phe Asn Thr 435 44a tct cgc caa cat tta gcc tta tta tta cta gag cct gat gat caa Ser Arg Gln His Leu Ala Leu Leu Leu Leu Glu Pro Asp Asp Gln 456t atc aac tta gtt aat act ttc agc cat tat atc act ggc gca Arg Ile Asn Leu Val Asn Thr Phe Ser His Tyr Ile Thr Gly Ala465 478g caa gtg cca ccg ggt ggt aaa gat ggt tta cgc cct gat ggt Thr Gln Val Pro Pro Gly Gly Lys Asp Gly Leu Arg Pro Asp Gly 485 49a gca tgg cga cat gaa ggc aac tat ccg ggc tac tct ttc cca gcc Ala Trp Arg His Glu Gly Asn Tyr Pro Gly Tyr Ser Phe Pro Ala 55aa aat gcc tct cag ctt att tat tta tta cgc gat aca cca ttt Lys Asn Ala Ser Gln Leu Ile Tyr Leu Leu Arg Asp Thr Pro Phe 5525tca gtg ggt gaa agt ggt tgg aat aac ctg aaa aaa gcg atg gtt tca Val Gly Glu Ser Gly Trp Asn Asn Leu Lys Lys Ala Met Val Ser 534g atc tac agt aat cca gaa gtt gga tta ccg ctt gca gga aga Trp Ile Tyr Ser Asn Pro Glu Val Gly Leu Pro Leu Ala Gly Arg545 556t ttt aac tca cct tcg tta aaa tca gtc gct caa ggc tat tac Pro Phe Asn Ser Pro Ser Leu Lys Ser Val Ala Gln Gly Tyr Tyr 565 57g ctt gcc atg tct gca aaa tca tcg cct gat aaa aca ctt gca tct Leu Ala Met Ser Ala Lys Ser Ser Pro Asp Lys Thr Leu Ala Ser 589t ctt gcg att agt gat aaa aca caa aat gaa tca act gct att Tyr Leu Ala Ile Ser Asp Lys Thr Gln Asn Glu Ser Thr Ala Ile 595 6tt gga gaa act att aca cca gcg tct tta cct caa ggt ttc tat gcc Gly Glu Thr Ile Thr Pro Ala Ser Leu Pro Gln Gly Phe Tyr Ala 662t ggc ggt gct ttt ggt att cat cgt tgg caa gat aaa atg gtg Asn Gly Gly Ala Phe Gly Ile His Arg Trp Gln Asp Lys Met Val625 634g aaa gct tat aac acc aat gtt tgg tca tct gaa att tat aac Leu Lys Ala Tyr Asn Thr Asn Val Trp Ser Ser Glu Ile Tyr Asn 645 65a gat aac cgt tat ggc cgt tac caa agt cat ggt gtc gct caa ata 2Asp Asn Arg Tyr Gly Arg Tyr Gln Ser His Gly Val Ala Gln Ile 667t aat ggc tcg cag ctt tca cag ggc tat cag caa gaa ggt tgg 2Ser Asn Gly Ser Gln Leu Ser Gln Gly Tyr Gln Gln Glu Gly Trp 675 68t tgg aat aga atg caa ggg gca acc act att cac ctt cct ctt aaa 2Trp Asn Arg Met Gln Gly Ala Thr Thr Ile His Leu Pro Leu Lys 69ta gac agt cct aaa cct cat acc tta atg caa cgt gga gag cgt 2Leu Asp Ser Pro Lys Pro His Thr Leu Met Gln Arg Gly Glu Arg77ga ttt agc gga aca tca tcc ctt gaa ggt caa tat ggc atg atg gca 22he Ser Gly Thr Ser Ser Leu Glu Gly Gln Tyr Gly Met Met Ala 725 73c gat ctt att tat ccc gcc aat ctt gag cgt ttt gat cct aat ttc 2256Phe Asp Leu Ile Tyr Pro Ala Asn Leu Glu Arg Phe Asp Pro Asn Phe 745g aaa aag agt gta tta gcc gct gat aat cac tta att ttt att 23la Lys Lys Ser Val Leu Ala Ala Asp Asn His Leu Ile Phe Ile 755 76t agc aat ata aat agt agt gat aaa aat aaa aat gtt gaa acg acc 2352Gly Ser Asn Ile Asn Ser Ser Asp Lys Asn Lys Asn Val Glu Thr Thr 778c caa cat gcc att act cca aca tta aat acc ctt tgg att aat 24he Gln His Ala Ile Thr Pro Thr Leu Asn Thr Leu Trp Ile Asn785 79aa aag ata gaa aac atg cct tat caa aca aca ctt caa caa ggt 2448Gly Gln Lys Ile Glu Asn Met Pro Tyr Gln Thr Thr Leu Gln Gln Gly 88gg tta att gat agc aat ggc aat ggt tac tta att act caa gca 2496Asp Trp Leu Ile Asp Ser Asn Gly Asn Gly Tyr Leu Ile Thr Gln Ala 823a gta aat gta agt cgc caa cat cag gtt tca gcg gaa aat aaa 2544Glu Lys Val Asn Val Ser Arg Gln His Gln Val Ser Ala Glu Asn Lys 835 84t cgc caa ccg aca gaa gga aac ttt agc tcg gca tgg atc gat cac 2592Asn Arg Gln Pro Thr Glu Gly Asn Phe Ser Ser Ala Trp Ile Asp His 856t cgc ccc aaa gat gcc agt tat gag tat atg gtc ttt tta gat 264r Arg Pro Lys Asp Ala Ser Tyr Glu Tyr Met Val Phe Leu Asp865 878a cct gaa aaa atg gga gag atg gca caa aaa ttc cgt gaa aat 2688Ala Thr Pro Glu Lys Met Gly Glu Met Ala Gln Lys Phe Arg Glu Asn 885 89t ggg tta tat cag gtt ctt cgt aag gat aaa gac gtt cat att att 2736Asn Gly Leu Tyr Gln Val Leu Arg Lys Asp Lys Asp Val His Ile Ile 99at aaa ctc agc aat gta acg gga tat gcc ttt tat cag cca gca 2784Leu Asp Lys Leu Ser Asn Val Thr Gly Tyr Ala Phe Tyr Gln Pro Ala 9925tca att gaa gac aaa tgg atc aaa aag gtt aat aaa cct gca att gtg 2832Ser Ile Glu Asp Lys Trp Ile Lys Lys Val Asn Lys Pro Ala Ile Val 934t cat cga caa aaa gac act ctt att gtc agt gca gtt aca cct 288r His Arg Gln Lys Asp Thr Leu Ile Val Ser Ala Val Thr Pro945 956a aat atg act cgc caa aaa gca gca act cct gtc acc atc aat 2928Asp Leu Asn Met Thr Arg Gln Lys Ala Ala Thr Pro Val Thr Ile Asn 965 97c acg att aat ggc aaa tgg caa tct gct gat aaa aat agt gaa gtg 2976Val Thr Ile Asn Gly Lys Trp Gln Ser Ala Asp Lys Asn Ser Glu Val 989t cag gtt tct ggt gat aac act gaa ctg acg ttt acg agt tac 3Tyr Gln Val Ser Gly Asp Asn Thr Glu Leu Thr Phe Thr Ser Tyr 995 gt att cca caa gaa atc aaa ctc tcg cca ctc cct tga 3Gly Ile Pro Gln Glu Ile Lys Leu Ser Pro Leu Pro 2Proteus vulgaris 2Met Pro Ile Phe Arg Phe Thr Ala Leu Ala Met Thr Leu Gly Leu Leula Pro Tyr Asn Ala Met Ala Ala Thr Ser Asn Pro Ala Phe Asp 2Pro Lys Asn Leu Met Gln Ser Glu Ile Tyr His Phe Ala Gln Asn Asn 35 4 Leu Ala Asp Phe Ser Ser Asp Lys Asn Ser Ile Leu Thr Leu Ser 5Asp Lys Arg Ser Ile Met Gly Asn Gln Ser Leu Leu Trp Lys Trp Lys65 7Gly Gly Ser Ser Phe Thr Leu His Lys Lys Leu Ile Val Pro Thr Asp 85 9 Glu Ala Ser Lys Ala Trp Gly Arg Ser Ser Thr Pro Val Phe Ser Trp Leu Tyr Asn Glu Lys Pro Ile Asp Gly Tyr Leu Thr Ile Asp Gly Glu Lys Leu Ile Ser Thr Ser Glu Ala Gln Ala Gly Phe Lys Lys Leu Asp Phe Thr Gly Trp Arg Ala Val Gly Val Ser Leu Asn Asn Asp Leu Glu Asn Arg Glu Met Thr Leu Asn Ala Thr Asn Thr Ser Asp Gly Thr Gln Asp Ser Ile Gly Arg Ser Leu Gly Ala Lys Val Ser Ile Arg Phe Lys Ala Pro Ser Asn Val Ser Gln Gly Glu Ile 2le Asp Arg Ile Met Phe Ser Val Asp Asp Ala Arg Tyr Gln Trp 222p Tyr Gln Val Lys Thr Arg Leu Ser Glu Pro Glu Ile Gln Phe225 234n Val Lys Pro Gln Leu Pro Val Thr Pro Glu Asn Leu Ala Ala 245 25e Asp Leu Ile Arg Gln Arg Leu Ile Asn Glu Phe Val Gly Gly Glu 267u Thr Asn Leu Ala Leu Glu Glu Asn Ile Ser Lys Leu Lys Ser 275 28p Phe Asp Ala Leu Asn Ile His Thr Leu Ala Asn Gly Gly Thr Gln 29rg His Leu Ile Thr Asp Lys Gln Ile Ile Ile Tyr Gln Pro Glu33sn Leu Asn Ser Gln Asp Lys Gln Leu Phe Asp Asn Tyr Val Ile Leu 325 33y Asn Tyr Thr Thr Leu Met Phe Asn Ile Ser Arg Ala Tyr Val Leu 345s Asp Pro Thr Gln Lys Ala Gln Leu Lys Gln Met Tyr Leu Leu 355 36t Thr Lys His Leu Leu Asp Gln Gly Phe Val Lys Gly Ser Ala Leu 378r Thr His His Trp Gly Tyr Ser Ser Arg Trp Trp Tyr Ile Ser385 39eu Leu Met Ser Asp Ala Leu Lys Glu Ala Asn Leu Gln Thr Gln 44yr Asp Ser Leu Leu Trp Tyr Ser Arg Glu Phe Lys Ser Ser Phe 423t Lys Val Ser Ala Asp Ser Ser Asp Leu Asp Tyr Phe Asn Thr 435 44u Ser Arg Gln His Leu Ala Leu Leu Leu Leu Glu Pro Asp Asp Gln 456g Ile Asn Leu Val Asn Thr Phe Ser His Tyr Ile Thr Gly Ala465 478r Gln Val Pro Pro Gly Gly Lys Asp Gly Leu Arg Pro Asp Gly 485 49r Ala Trp Arg His Glu Gly Asn Tyr Pro Gly Tyr Ser Phe Pro Ala 55ys Asn Ala Ser Gln Leu Ile Tyr Leu Leu Arg Asp Thr Pro Phe 5525Ser Val Gly Glu Ser Gly Trp Asn Asn Leu Lys Lys Ala Met Val Ser 534p Ile Tyr Ser Asn Pro Glu Val Gly Leu Pro Leu Ala Gly Arg545 556o Phe Asn Ser Pro Ser Leu Lys Ser Val Ala Gln Gly Tyr Tyr 565 57p Leu Ala Met Ser Ala Lys Ser Ser Pro Asp Lys Thr Leu Ala Ser 589r Leu Ala Ile Ser Asp Lys Thr Gln Asn Glu Ser Thr Ala Ile 595 6he Gly Glu Thr Ile Thr Pro Ala Ser Leu Pro Gln Gly Phe Tyr Ala 662n Gly Gly Ala Phe Gly Ile His Arg Trp Gln Asp Lys Met Val625 634u Lys Ala Tyr Asn Thr Asn Val Trp Ser Ser Glu Ile Tyr Asn 645 65s Asp Asn Arg Tyr Gly Arg Tyr Gln Ser His Gly Val Ala Gln Ile 667r Asn Gly Ser Gln Leu Ser Gln Gly Tyr Gln Gln Glu Gly Trp 675 68p Trp Asn Arg Met Gln Gly Ala Thr Thr Ile His Leu Pro Leu Lys 69eu Asp Ser Pro Lys Pro His Thr Leu Met Gln Arg Gly Glu Arg77ly Phe Ser Gly Thr Ser Ser Leu Glu Gly Gln Tyr Gly Met Met Ala 725 73e Asp Leu Ile Tyr Pro Ala Asn Leu Glu Arg Phe Asp Pro Asn Phe 745a Lys Lys Ser Val Leu Ala Ala Asp Asn His Leu Ile Phe Ile 755 76y Ser Asn Ile Asn Ser Ser Asp Lys Asn Lys Asn Val Glu Thr Thr 778e Gln His Ala Ile Thr Pro Thr Leu Asn Thr Leu Trp Ile Asn785 79ln Lys Ile Glu Asn Met Pro Tyr Gln Thr Thr Leu Gln Gln Gly 88rp Leu Ile Asp Ser Asn Gly Asn Gly Tyr Leu

Ile Thr Gln Ala 823s Val Asn Val Ser Arg Gln His Gln Val Ser Ala Glu Asn Lys 835 84n Arg Gln Pro Thr Glu Gly Asn Phe Ser Ser Ala Trp Ile Asp His 856r Arg Pro Lys Asp Ala Ser Tyr Glu Tyr Met Val Phe Leu Asp865 878r Pro Glu Lys Met Gly Glu Met Ala Gln Lys Phe Arg Glu Asn 885 89n Gly Leu Tyr Gln Val Leu Arg Lys Asp Lys Asp Val His Ile Ile 99sp Lys Leu Ser Asn Val Thr Gly Tyr Ala Phe Tyr Gln Pro Ala 9925Ser Ile Glu Asp Lys Trp Ile Lys Lys Val Asn Lys Pro Ala Ile Val 934r His Arg Gln Lys Asp Thr Leu Ile Val Ser Ala Val Thr Pro945 956u Asn Met Thr Arg Gln Lys Ala Ala Thr Pro Val Thr Ile Asn 965 97l Thr Ile Asn Gly Lys Trp Gln Ser Ala Asp Lys Asn Ser Glu Val 989r Gln Val Ser Gly Asp Asn Thr Glu Leu Thr Phe Thr Ser Tyr 995 ly Ile Pro Gln Glu Ile Lys Leu Ser Pro Leu Pro 3tificial SequenceDescription of Artificial Sequence Synthetic peptide 3Ala Thr Ser Asn Pro Ala Phe Asp Pro Lys Asn Leu Met Gln Ser Gluyr42ificial SequenceDescription of Artificial Sequence Synthetic primer 4ctaatgggtt attttgtgca a 2Artificial SequenceDescription of Artificial Sequence Synthetic primer 5gcnacnucna ayccngc AArtificial SequenceDescription of Artificial Sequence Synthetic primer 6gcnacnagya ayccngc AArtificial SequenceDescription of Artificial Sequence Synthetic primer 7uacguyagnc uyuadau AArtificial SequenceDescription of Artificial Sequence Synthetic primer 8uacguyucrc uyuadau AArtificial SequenceDescription of Artificial Sequence Synthetic probe 9catttgatcc taaaaatctg atgca 25Artificial SequenceDescription of Artificial Sequence Synthetic primer cagca atcctgca NAArtificial SequenceDescription of Artificial Sequence Synthetic primer ttcgg actgcatcag 2AArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide cc agc aat cct gca ttt gat cct aaa aat ctg atg cag tcc gaa 48Ala Thr Ser Asn Pro Ala Phe Asp Pro Lys Asn Leu Met Gln Ser Glua 53Ile TyrArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide ttcgg actgcatcag atttttagga tcaaatgcag gattgctggt tgc 53AProteus vulgaris cactc aatcattaaa tttaggcaca acgatgggct atcagcgtta tgacaaattt 6ggac gcattggttt cactgttagc cagcgtttct aaggagaaaa ataatgccga 22Phaseolus vulgaris aaatt taatgaagga cgcattggtt tcactgttag ccagcgtttc taaggagaaa 6NAProteus vulgarisCDS(296)..(4agactgctt atggcaaatt aaccccctct cttaatcttc gttattcaaa gatattgcag 6atga tatcaatcaa cgccacagcc ttacctattt taatacaggg ggaagtacct tattaa aggaaatacc gttggtggtg acattattag tgcggaatta ggtgcaaatc tatcac tcaatcatta aatttaggca caacgatggg ctatcagcgt tatgacaaat 24aagg acgcattggt ttcactgtta gccagcgttt ctaaggagaa aaata atg 298 Met a ttt cgt ttt act gca ctt gca atg aca ttg ggg cta tta tca 346Pro Ile Phe Arg Phe Thr Ala Leu Ala Met Thr Leu Gly Leu Leu Ser 5 g cct tat aac gcg atg gca gcc acc agc aat cct gca ttt gat cct 394Ala Pro Tyr Asn Ala Met Ala Ala Thr Ser Asn Pro Ala Phe Asp Pro 2aaa aat 4sn 35Proteus vulgaris ro Ile Phe Arg Phe Thr Ala Leu Ala Met Thr Leu Gly Leu Leula Pro Tyr Asn Ala Met Ala Ala Thr Ser Asn Pro Ala Phe Asp 2Pro Lys Asn 35

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