You’re using a public version of DrugPatentWatch with 5 free searches available | Register to unlock more free searches. CREATE FREE ACCOUNT

Last Updated: March 28, 2024

Claims for Patent: 5,177,017


✉ Email this page to a colleague

« Back to Dashboard


Summary for Patent: 5,177,017
Title: Molecular cloning of the genes responsible for collagenase production from Clostridium histolyticum
Abstract:Genetically engineered E. coli carry vectors containing inserts that code for Clostridium histolyticum collagenase. These inserts code for: (a) a form of collagenase having a molecular weight of about 68,000 daltons in the essential absence of larger forms of collagenase; (b) the 68 kd form of collagenase and a fusion polypeptide consisting of the collagenase protein fused to at least a portion of the .beta.-galactosidase protein of E. coli; or (3) the 68 kd form of collagenase and polypeptides of molecular weight of from above about 68,000 daltons to about 100,000 daltons and having the enzymatic activity of C. histolyticum collagenase as indicated by digestion of .sup.3 H-acetylated collagen and by specific inhibition by 1,10-phenanthroline plus EDTA. The collagenase genes in the transformed E. coli are expressed efficiently in the transformed cells to yield enzymatically active and immunologically cross-reactive collagenase. In particular, the 68 kd form of collagenase is resistant to autocatalytic degradation and is stable to long-term storage. Genetically engineered collagenase, especia The research reported in this application in support of the invention herein received assistance from a grant provided by the Department of Health & Human Services, Public Health Service, National Institute of Diabetes, Digestive and Kidney Disease, Grant No. N43-DK-8-2239.
Inventor(s): Lin; Hun-Chi (Los Angeles, CA), Lei; Shau-Ping (Los Angeles, CA)
Assignee: Trigen, Inc. (Santa Monica, CA)
Application Number:07/498,919
Patent Claims:1. An isolated and purified recombinant DNA fragment consisting essentially of DNA derived from Clostridium histolyticum coding solely for a polypeptide having the enzymatic activity of C. histolyticum collagenase, the polypeptide having a molecular weight of about 68,000 daltons.

2. The DNA fragment of claim 1 capable of being transcribed to yield mRNA, the mRNA being capable of being translated to yield a polypeptide of about 68,000 daltons molecular weight and having collagenase activity, the polypeptide being distinguishable by the heterologous expression of the sequence of claim 3 from the endogenous production by C. histolyticum of mutliple forms of collagenase by the essential absence of those forms of C. histolyticum collagenase having molecular weights of above about 70,000 daltons determined by the expression of the C. histolyticum genomic coding sequence.

3. The DNA fragment of claim 1 capable of being transcribed to yield mRNA, the mRNA being capable of being translated to yield a peptide displaying the antigenicity of C. histolyticum collagenease, the polypeptide being distinguishable by the heterologous expression of the sequence of claim 3 from the endogenous production by C. histolyticum of multiple forms of collagenase by the essential absence of those forms of C. histolyticum collagenase having molecular weights of above about 70,000 daltons determined by the expression of the C. histolyticum genomic coding sequence.

4. The DNA fragment of claim 2 wherein the DNA derived from C. histolyticum contains a promoter within the inserted Clostridium DNA such that the DNA can be transcribed under control of the promoter to yield mRNA capable of being translated to yield the polypeptide of about 68,000 daltons without the functioning of a promoter external to the DNA derived from C. histolyticum.

5. A DNA fragment comprising the DNA fragment of claim 2 fused contiguously to a transcription-effecting DNA sequence selected from the group consisting of Escherichia coli lac promoter, E. coli trp promoter, bacteriophage .lambda. P.sub.L promoter, and tac promoter such that the transcription-effecting sequence can effect transcription of the DNA derived from C. histolyticum.

6. A DNA fragment comprising the DNA fragment of claim 4 fused contiguously at least a portion of the structural gene for E. coli .beta.-galactosidase, the portion of the structural gene for .beta.-galactosidase being operatively linked to a lac promoter such that:

(i) the DNA derived from Clostridium can be transcribed under control of the promoter located within the Clostridium DNA to yield mRNA capable of being translated to yield the polypeptide of about 68,000 daltons without the functioning of a promoter external to the DNA derived from C. histolyticum; and

(ii) the Clostridium DNA fused to the DNA sequence comprising at least a portion of the structural gene for E. coli .beta.-galactosidase can be transcribed under control of the lac promoter to yield mRNA capable of being translated to yield a fusion polypeptide of molecular weight greater than 70,000 daltons displaying the antigenicity of C. histolyticum collagenase and containing at least a portion of the amino acid sequence of E. coli .beta.-galactosidase.

7. A vector comprising the DNA fragment of claim 4 incorporated into a plasmid capable of stably transforming E. coli host cells selected from the group of plasmids consisting of those with both a drug resistance marker and a replication origin.

8. The vector of claim 7 wherein the plasmid is generated in vivo by rescue of .lambda.zap DNA containing the inserted Clostridium DNA by M13 helper phage.

9. A vector comprising the DNA fragment of claim 5 incorporated into a plasmid capable of stably transforming E. coli host cells selected from the group of plasmids consisting of those with both a drug resistance marker and a replication origin.

10. The vector of claim 9 wherein the plasmid is generated in vivo by rescue of .lambda.zap DNA containing the inserted Clostridium DNA by M13 helper phage.

11. A vector comprising the DNA fragment of claim 8 incorporated into a plasmid capable of stably transforming E. coli host cells selected from the group of plasmids consisting of those with both a drug resistance marker and a replication origin.

12. The vector of claim 11 wherein the plasmid is generated in vivo by rescue of .lambda.zap DNA containing the inserted Clostridium DNA by M13 helper phage.

13. An isolated and purified recombinant DNA fragment comprising:

(a) a Clostridium histolyticum structural gene encoding C. histolyticum collagenase and capable of being transcribed to yield mRNA capable of being translated to yield polypeptides of molecular weight of from about 70,000 dalonts to about 100,000 daltons and having the enzymatic activity of C. histolyticum collagenase; and

(b) an internal promoter located within the structural gene of (a) and operatively linked to a Clostridium DNA sequence comprising a portion of the structural gene of (a) and capable of being transcribed to yield mRNA capable of being translated to yield a polypeptide of about 68,000 daltons molecular weight, the polypeptide having collagenase activity, the polypeptide being distinguishable by the heterologous expression of the sequence of claim 3 from the endogenous production by C. histolyticum of multiple forms of collagenase by the essential absence of those forms of C. histolyticum collagenease having molecular weights of above about 70,000 daltons determined by the expression of the C. histolyticum genomic coding sequence; the internal promoter being positioned such that the DNA sequence of (b) can be translated independently of the structural gene of (a).

14. A DNA fragment comprising the DNA fragment of claim 13 fused contiguously to a transcription-effecting DNA sequence selected from the group consisting of E. coli lac promoter, E. coli trp promoter, bacteriophage .lambda. P.sub.L promoter, and tac promoter such that the transcription-effecting sequence can effect transcription of the structural gene of (a).

15. The DNA of claim 14 wherein the C. histolyticum structural gene of (a) and DNA sequence of (b) are capable of being alternatively and colinearly expressed to produce C. histolyticum collagenase having molecular weights ranging from about 68,000 daltons to about 100,000 daltons.

16. The DNA fragment of claim 15 wherein the internal promoter of (b) is capable of coding for and being read as an internal amino acid sequence of the structural gene of (a).

17. A vector comprising the DNA fragment of claim 14 incorporated into a plasmid capable of stably transforming E. coli host cells selected from the group of plasmids consisting of those with both a drug resistance marker and a replication origin.

18. The vector of claim 17 wherein the plasmid is generated in vivo by rescue of .lambda.zap DNA containing the inserted Clostridium DNA by M13 helper phage.

19. E. coli host cells transformed with the vector of claim 7 and producing a polypeptide of about 68,000 daltons having collagenase activity and distinguishable by the heterologous expression of the sequence of claim 3 from the endogenous production by C. histolyticum of multiple forms of collagenase by the essential absence of those forms of C. histolyticum collagenase having molecular weights of above about 70,000 daltons determined by the expression of the C. histolyticum genomic coding sequence, the polypeptide being sufficiently resistant to autocatalytic degradation that it is substantially stable on storage for about 2 days at about 25.degree. C.

20. E. coli host cells transformed with the vector of claim 9 and producing a polypeptide of about 68,000 daltons having collagenase activity and distinguishable by the heterologous expression of the sequence of claim 3 from the endogenous production by C. histolyticum of multiple forms of collagenase by the essential absence of those forms of C. histolyticum collagenase having molecular weights of above about 70,000 daltons determined by the expression of the C. histolyticum genomic coding sequence, the polypeptide being sufficiently resistant to autocatalytic degradation that it is substantially stable on storage for about 2 days at about 25.degree. C.

21. E. coli host cell transformed with the vector of claim 11 and producing:

(i) a polypeptide of about 68,000 daltons having collagenase activity and distinguishable by the heterologous expression of the sequence of claim 3 from the endogenous production by C. histolyticum of multiple forms of collagenase by the essential absence of those forms of C. histolyticum collagenase having molecular weights of above about 70,000 daltons determined by the expression of the C. histolyticum genomic coding sequence, the polypeptide being sufficiently resistant to autocatalytic degradation that it is substantially stable on storage for about 2 days at about 25.degree. C.; and (ii) a fusion polypeptide of molecular weight greater than 70,000 daltons displaying the antigenicity of C. histolyticum collagenase and containing at a least a portion of the amino acid sequence of E. coli galactosidase.

22. E. coli host cells transformed with the vector of claim 17 and producing two forms of collagenase:

(i) a polypeptide of about 68,000 daltons having collagenase activity and distinguishable by the heterologous expression of the sequence of claim 3 from the endogenous production by C. histolyticum of multiple forms of collagenase by the essential absence of those forms of C. histolyticum collagenase having molecular weights of above about 70,000 daltons determined by the expression of the C. histolyticum genomic coding sequence, the polypeptide being sufficiently resistant to autocatalytic degradation that it is substantially stable on storage for about 2 days at about 25.degree. C.; and

(ii) polypeptides with molecular weights of from above 70,000 daltons to about 100,000 daltons and having collagenase activity.

23. The E. coli host cells of claim 22 wherein the polypeptide of about 68,000 daltons comprises greater than 50% by weight of the total polypeptides produced that have collagenase activity.

Make Better Decisions: Try a trial or see plans & pricing

Drugs may be covered by multiple patents or regulatory protections. All trademarks and applicant names are the property of their respective owners or licensors. Although great care is taken in the proper and correct provision of this service, thinkBiotech LLC does not accept any responsibility for possible consequences of errors or omissions in the provided data. The data presented herein is for information purposes only. There is no warranty that the data contained herein is error free. thinkBiotech performs no independent verification of facts as provided by public sources nor are attempts made to provide legal or investing advice. Any reliance on data provided herein is done solely at the discretion of the user. Users of this service are advised to seek professional advice and independent confirmation before considering acting on any of the provided information. thinkBiotech LLC reserves the right to amend, extend or withdraw any part or all of the offered service without notice.