Process for producing heterologous polypeptides

• US 5,411,873 A
• Filed: 08/11/1992
• Issued: 05/02/1995
• Est. Priority Date: 05/29/1984
• Status: Expired due to Term

• Current Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Wells, James A., Powers, David B., Power, Scott D., Yansura, Daniel G., Adams, Robin M.
• Primary Examiner(s): Martinell, James
• Application Number:
  US07/928,697
• Time in Patent Office: 994 Days
• Field of Search: 435/69.1, 435/69.7, 435/172.1, 435/172.3, 435/199, 435/212, 435/320.1, 435/69.8, 435/252.31, 935/10, 935/14, 935/51, 935/48, 935/74, 536/23.4, 536/23.7
• US Class Current: 435/69.1
• CPC Class Codes: C11D 3/386 Preparations containing enzymes , e.g. protease or amylase
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/75 for Bacillus
• CPC Class Codes: C12N 9/54 bacteria being Bacillus
• CPC Class Codes: C12R 2001/07 Bacillus

Abstract

Processes for producing various heterologous polypeptides which when expressed are either incorrectly processed and hence asssociated with the surface of the host cell or are not processed to mature form. More specifically, processes for the production of heterologous non-human carbonyl hydrolases expressed either in host cells incapable of producing enzymatically active endoprotease or host cells deficient in enzymatically active extracellular endoprotease are disclosed. Such non-human carbonyl hydrolases generally are incapable of autoproteolytic maturation and become associated with the surface of expression hosts which are deficient in enzymatically active extracellular endoprotease. Processes for preparing non-human carbonyl hydrolase and heterologous polypeptides which are expressed as part of a fusion polypeptide are also disclosed, as well as non-human carbonyl hydrolases which are substantially free of the host cell membrane with which they are normally associated.

Claims

1. A process for producing a subtilisin which has an amino acid substitution in one or more amino acid residues of the catalytic triad of wild-type subtilisin and is incapable of autoproteolytic maturation, wherein said process comprises:

a) expressing a prepro form of said subtilisin in a host cell and transporting it to the surface of said host cell, wherein said host cell is incapable of releasing said subtilisin from the surface of said host cell, and wherein the prosequence of said prepro form of said subtilisin comprises the prosequence of a subtilisin; and

b) contacting said host cell with an enzymatically active subtilisin to cleave said subtilisin from said prosequence to release said subtilisin from the surface of said host cell.

2. The process of claim 1 wherein said prepro form of said subtilisin which has an amino acid substitution in one or more amino acid residues of the catalytic triad and is incapable of autoproteolytic maturation contains a signal sequence capable of transporting said subtilisin to the surface of said host cell.

3. The process of claim 2 wherein said signal sequence is the signal sequence of subtilisin.

4. The process of claim 1 wherein said prosequence of said prepro form of said subtilisin which has an amino acid substitution in one or more amino acid residues of the catalytic triad and is incapable of autoproteolytic maturation comprises the prosequence of an enzymatically active subtilisin.

5. The process of claim 1 wherein said contacting is achieved by co-culturing an enzymatically active subtilisin secreting organism with said host cells.

6. The process of claim 1 wherein said contacting is achieved by adding enzymatically active exogenous subtilisin to a culture of said host cells.

7. The process of claim 1 wherein said mutant subtilisin has an amino acid substitution in the catalytic triad at position 221.

8. The process of claim 1 wherein said mutant subtilisin has an amino acid substitution in the catalytic triad at position 32.

9. The process of claim 1 wherein said mutant subtilisin has an amino acid substitution in the catalytic triad at position 64.

10. The process of claim 1 wherein said mutant subtilisin is selected from the group consisting of Ser(221)→

AlaΔ

166 and Asp(32)→

Asn.

11. The process of claim 1 wherein said enzymatically active subtilisin is Ala(48)→

Arg.

12. The process of claim 1 wherein said enzymatically active subtilisin is a thermolabile subtilisin.

13. The process of claim 12 wherein said thermolabile subtilisin comprises Met(222)→

Asp.

14. The process of claim 12 wherein said host cells comprises Bacillus subtilis strains which have the genotype apr-npr-.

15. The process of claim 1 wherein said enzymatically active subtilisin is produced by said host cell.

16. The process of claim 15 wherein said host cells comprise Bacillus strains which have the genotype apr-npr+,

17. The process of claim 15 wherein said host cells comprise Bacillus strains which have the genotype apr+npr-.

18. The process of claim 15 wherein said host cells comprise Bacillus strains which have the genotype apr+npr+.

19. A process for producing a polypeptide in a host cell comprising:

a) producing a fusion polypeptide having an amino-terminal first sequence comprising the prosequence of a subtilisin and a carboxy terminal second sequence comprising a desired polypeptide having a sequence other than that of wild-type subtilisin, wherein said fusion polypeptide is capable of being cleaved by an enzymatically active subtilisin at the junction of said first and second sequences or within said first sequence; and

b) contacting said fusion polypeptide with said enzymatically active subtilisin to cleave said fusion polypeptide at said junction.

20. The process of claim 19 wherein said contacting step is achieved by co-culturing said host cell and an organism which secretes an enzymatically active subtilisin.

21. The process of claim 19 wherein said desired polypeptide is Δ

166 subtilisin.

22. The process of claim 19 wherein said contacting is achieved by adding enzymatically active exogenous subtilisin to a culture of said host cells.