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).fwdarw.Ala.DELTA.166 and Asp(32).fwdarw.Asn.
- 11. The process of claim 1 wherein said enzymatically active subtilisin is Ala(48).fwdarw.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).fwdarw.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 .DELTA.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.
Parent Case Info
This application is a continuation of U.S. application Ser. No. 07/488,433, filed Feb. 27, 1990, now abandoned, which is a continuation of 06/846,627, filed Apr. 1, 1986, now abandoned, which is a continuation in part of U.S. application Ser. No. 06/614,612, filed May 29, 1984, which issued as U.S. Pat. No. 4,760,025 on Jul. 26, 1988, each of which is incorporated herein by reference.
US Referenced Citations (6)
Foreign Referenced Citations (3)
Number |
Date |
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0130756 |
Jan 1985 |
EPX |
0158981 |
Apr 1985 |
EPX |
0161937 |
Nov 1985 |
EPX |
Non-Patent Literature Citations (3)
Entry |
Zaghloul et al, Journal of Bacteriology 164 (2) pp. 550-555 (1985). |
Vasantha et al, Journal of Bacteriology 159 (3) pp. 811-819 (1984). |
Vasantha et al, Journal of Bacterology 165 (3) pp. 837-842 (1986). |
Continuations (2)
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Number |
Date |
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Parent |
488433 |
Feb 1990 |
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Parent |
846627 |
Apr 1986 |
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Continuation in Parts (1)
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Number |
Date |
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Parent |
614612 |
May 1984 |
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