Designer proteases

• US 6,383,775 B1
• Filed: 03/11/1999
• Issued: 05/07/2002
• Est. Priority Date: 09/11/1996
• Status: Expired due to Fees

• Current Assignee: Interleukin Genetics Incorporated (Orig3n, Inc.)
• Original Assignee: Interleukin Genetics Incorporated (Orig3n, Inc.)
• Inventors: Vitovski, Srdjan, Sayers, Jon R., Duff, Gordon W.
• Primary Examiner(s): Venkat, Jyothsna
• Assistant Examiner(s): FRIEND, TOMAS H F
• Application Number:
  US09/266,498
• Time in Patent Office: 1,153 Days
• Field of Search: 435/7.1, 435/69.7, 435/69.8, 435/70.1, 435/69.1, 530/350, 530/370, 530/371, 530/380, 530/381
• US Class Current: 435/69.1
• CPC Class Codes: C12N 9/52 derived from bacteria or Archaea

Abstract

The invention provides a method for engineering and selecting an active protease able to cleave a user-defined target amino acid sequence. The method is useful for designing proteases for medical therapeutics and industrial applications.

Claims

1. A method for obtaining a target specific protease, comprising the steps of:

a) providing a DNA vector encoding a modified self-secreting endoprotease, wherein said endoprotease is comprised of a signal peptide sequence, an endoprotease catalytic domain, a target cleavage site of a target protein and a carboxy-terminal domain;

b) obtaining a library of said vectors, wherein the DNA encoding the endoprotease catalytic domain has been mutagenized; and

c) isolating from said library a vector that expresses a target specific protease that is able to cleave the target cleavage site of said target protein.

2. The method of claim 1, wherein the endoprotease catalytic domain is an IgA endoprotease catalytic domain.

3. The method of claim 1, wherein said target cleavage site is identical to a sequence present in a target protein selected from the group consisting of:

a cytokine, a cytokine receptor, a clotting factor, a gene product of an oncogene, a gene product of an anti-oncogene, a mutant protein that causes a genetic disorder, a viral protein, a bacterial protein, and a polypeptide-containing toxin or environmental or industrial pollutant.

4. The method of claim 1, wherein said target protein is overexpressed in humans having a disease.

5. The method of claim 1 or 4, wherein said target protein is selected from the group consisting of HIV-1 protease, HIV-1 reverse transcriptase, HIV-1 integrase, HIV-1 gp120, HIV-1 gp41, urokinase-type plasminogen activator, tissue-type plasminogen activator, neutrophil elastase, argingipain, von Willebrand factor, p53 and Her-2/neu.

6. The method of claim 1, wherein the library of vectors in step (b) is obtained by site directed mutagenesis or Stemmer gene shuffling.

7. The method of claim 1 or 4, wherein the library obtained in step (b) is transferred to a bacterial host cell which can express the protease products.

8. The method of claim 1 or 4, further comprising step (d) wherein steps (b) and (c) are repeated until the desired endoprotease catalytic domain specificity is obtained.

9. The method of claim 1, wherein the isolation of said vector from said library comprises a negative selection wherein a cell expressing IgA protease on its surface is depleted.

10. The method of claim 1, wherein the isolation of said vector from said library comprises a positive selection.

11. The method of claim 1, wherein the isolation of said vector from said library comprises detection of the endoprotease catalytic domain which is liberated upon cleavage of said target cleavage site.

12. The method of claim 1, further including the step of isolating cells producing a protein comprising the mutagenized endoprotease catalytic domain which cleaves said target cleavage site of said target protein.

13. The method of claim 1, wherein said modified self-secreting enoprotease further comprises an affinity tag which can be used in a positive selection procedure to select bacteria containing a vector encoding a protease catalytic domain which cleaves the target cleavage site and unmasks the affinity tag.

14. The method of claim 13, wherein the affinity tag is able to bind to an immobilized ligand.

15. A modified self-secreting endoprotease which has been engineered to cleave a user-defined target cleavage site, said endoprotease comprising:

(a) a signal peptide sequence,

(b) an endoprotease catalytic domain,

(c) a target cleavage site and

(d) a carboxy-terminal domain.

16. The modified self-secreting endoprotease of claim 15, further comprising an affinity tag.

17. The modified self-secreting endoprotease of claim 15, wherein said protease catalytic domain is a mutant protease catalytic domain which cleaves said target cleavage site.