Method for engineering proteases and protein kinases

• US 9,546,359 B2
• Filed: 12/19/2014
• Issued: 01/17/2017
• Est. Priority Date: 06/25/2012
• Status: Active Grant

• Current Assignee: Research & Development Foundation
• Original Assignee: Research & Development Foundation
• Inventors: Iverson, Brent, Georgiou, George, Taft, Joseph M., Yi, Li
• Primary Examiner(s): Carlson, Karen Cochrane
• Application Number:
  US14/576,766
• Publication Number:
  US 20150203834A1
• Time in Patent Office: 760 Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 2800/60 Vectors containing traps for, e.g. exons, promoters
• CPC Class Codes: C12N 2810/40 Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
• CPC Class Codes: C12N 9/12 transferring phosphorus containing groups, e.g. kinases (2.7)
• CPC Class Codes: C12N 9/60 from yeast

Abstract

Provided are methods for protein engineering, such as engineering proteases or kinases. The methods may utilize yeast display and/or ER sequestration of proteins or substrates. In some aspects, TEV proteases with altered substrate specificity, potency, and/or efficiency are provided.

Claims

1. A nucleic acid vector for engineering protease variants, wherein the nucleic acid vector encodes two separately expressed proteins, wherein the first protein is a first fusion protein comprising, in an N- to C-terminal direction:

(i) a first endoplasmic reticulum (ER) targeting sequence;

(ii) a surface expression sequence;

(iii) a first peptide sequence that is a counterselection substrate sequence for the enzyme of the second protein;

(iv) a first epitope tag sequence;

(v) a second peptide sequence that is a selection substrate sequence for the enzyme of the second fusion protein;

(vi) a second epitope tag sequence; and

(vii) a first endoplasmic reticulum (ER) retention sequence;

wherein the second protein is a protease or has protease activity.

2. The nucleic acid of claim 1, wherein the nucleic acid encoding the first fusion protein is operably linked to a first promoter;

and wherein the nucleic acid encoding the second protein is operably linked to a second promoter.

3. The nucleic acid of claim 2, wherein the first promoter and the second promoter are expressable in yeast.

4. The nucleic acid of claim 2, wherein the first promoter is Gal1 or Gal10.

5. The nucleic acid of claim 2, wherein the second promoter is Gal1 or Gal10.

6. The nucleic acid of claim 2, wherein the nucleic acid comprises one or more enhancers.

7. The nucleic acid of claim 1, wherein at least a portion of the first peptide is randomized.

8. The nucleic acid of claim 1, wherein the first peptide is a sequence that has no sequence identity with the native substrate of the protease.

9. The nucleic acid of claim 1, wherein the first peptide is a mutated native substrate of the protease.

10. The nucleic acid of claim 1, wherein at least a portion of the second peptide is randomized.

11. The nucleic acid of claim 1, wherein the second peptide is the native substrate of the protease.

12. The nucleic acid of claim 1, wherein the second protein is further defined as a second fusion protein, wherein the second fusion protein comprises, in an N- to C-terminal direction:

a second endoplasmic reticulum (ER) targeting sequence, a protease, and a second endoplasmic reticulum (ER) retention sequence.

13. The nucleic acid of claim 1, wherein the second protein is a protease, wherein the protease is not a fusion protein.

14. The nucleic acid of claim 1, wherein the protease is a human protease.

15. The nucleic acid of claim 14 wherein the protease is a TEV-protease, rTPA, a coagulation factor, factor 7, factor 9, human trypsin, a granzyme, a caspase, trypsin, human granzyme K, or a human caspase.

16. The nucleic acid of claim 1, wherein at least a portion of the protease is randomized.

17. The nucleic acid of claim 1, wherein the protease has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mutations, additions, or deletions as compared to the wild-type protease.

18. The nucleic acid of claim 1, wherein the first endoplasmic reticulum (ER) targeting sequence is MQLLRCFSIFSVIASVLA (SEQ ID NO:

3).

19. The nucleic acid of claim 1, wherein the first endoplasmic reticulum (ER) retention sequence is FEHDEL (SEQ ID NO:

4), KDEL (SEQ ID NO;

5), HDEL (SEQ ID NO;

6), or RDEL (SEQ ID NO;

7).

20. The nucleic acid of claim 1, wherein the nucleic acid further encodes a third epitope tag sequence.

21. The nucleic acid of claim 20, wherein the third epitope tag sequence is a hemagglutinin epitope tag.

22. The nucleic acid of claim 20, wherein the third epitope tag is comprised in the first fusion construct.

23. The nucleic acid of claim 20, wherein the third epitope tag is located between (ii) and (iii).

24. A cell comprising the nucleic acid vector of claim 1.

25. The cell of claim 24, wherein the cell is a yeast cell.

26. A method for producing a protease, comprising:

(i) expressing one or more nucleic acid of claim 1 in a plurality of cells;

(ii) purifying or separating cells based on the presence or absence of an antibody that selectively binds the first epitope tag sequence or the second epitope tag sequence, and

(iii) isolating or purifying the protease.

27. The method of claim 26, wherein the cell is a yeast cell.

28. The method of claim 26, wherein the nucleic acid further encodes a third epitope tag.

29. The method of claim 28, further comprising purifying cells that express the third epitope tag.

30. The method of claim 26, wherein the antibody is labeled with a fluorophore.

31. The method of claim 26, wherein the isolating or purifying the protease comprises FACS.

32. The method of claim 26, further comprising isolating the nucleic acid.

33. The method of claim 32, further comprising further randomizing a portion of the nucleic acid.

34. The method of claim 26, further comprising further characterizing the protease encoded by the nucleic acid.

35. The method of claim 34, further comprising repeating steps (i) and (ii).