Method for engineering proteases and protein kinases

• US 8,945,855 B2
• Filed: 06/25/2013
• Issued: 02/03/2015
• Est. Priority Date: 06/25/2012
• Status: Active Grant

• Current Assignee: Research & Development Foundation
• Original Assignee: Research & Development Foundation
• Inventors: Iverson, Brent L., Georgiou, George, Taft, Joseph M., Yi, Li
• Primary Examiner(s): Carlson, Karen Cochrane
• Application Number:
  US13/926,749
• Publication Number:
  US 20140017764A1
• Time in Patent Office: 588 Days
• Field of Search: 435/7.1, 435/194, 435/219, 435/254.2, 435/320.1
• US Class Current: 435/7.1
• 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 fusion proteins, wherein the first fusion protein comprise, 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 fusion 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 fusion protein comprises, in an N- to C-terminal direction;

(viii) a second endoplasmic reticulum (ER) targeting sequence;

(ix) an enzyme, wherein the enzyme is a protease; and

(x) a second endoplasmic reticulum (ER) retention sequence.

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 fusion 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 is unrelated to the native substrate or shares no or essentially 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 9, wherein the first peptide has 1, 2, 3, 4, or 5 mutations, additions, or deletions as compared to the native substrate of the protease.

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

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

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

14. The nucleic acid of claim 13 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.

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

16. 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.

17. The nucleic acid of claim 1, wherein the first endoplasmic reticulum (ER) targeting sequence and the second endoplasmic reticulum (ER) targeting sequence are MQLLRCFSIFSVIASVLA (SEQ ID NO:

3).

18. The nucleic acid of claim 1, wherein the first endoplasmic reticulum (ER) retention sequence and the second endoplasmic reticulum (ER) retention sequence are FEHDEL (SEQ ID NO:

4), KDEL (SEQ ID NO;

5), HDEL (SEQ ID NO;

6), or RDEL (SEQ ID NO;

7).

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

20. The nucleic acid of claim 19, wherein the third epitope tag sequence is a hemagglutinin epitope tag.

21. The nucleic acid of claim 19, wherein the third epitope tag is comprised in the first fusion construct.

22. The nucleic acid of claim 19, wherein the third epitope tag is located between (ii) and (iii).

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

24. The cell of claim 23, wherein the cell is a yeast cell.

25. 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.

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

27. The method of claim 25, wherein the nucleic acid further encodes a third epitope tag.

28. The method of claim 27, further comprising purifying cells that express the third epitope tag.

29. The method of claim 25, wherein the antibody is labeled with a fluorophore.

30. The method of claim 25, wherein the isolating or purifying the protease comprises FACS.

31. The method of claim 25, further comprising isolating the nucleic acid.

32. The method of claim 31, further comprising further randomizing a portion of the nucleic acid.

33. The method of claim 25, further comprising further characterizing the protease encoded by the nucleic acid.

34. The method of claim 33, further comprising repeating steps (i) and (ii).

35. A nucleic acid vector for engineering protease and substrate variants, wherein the nucleic acid vector encodes two separately expressed fusion proteins, wherein the first fusion protein comprises, in an N- to C-terminal direction:

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

(ii) a surface expression sequence;

(iii) a first epitope tag sequence;

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

(v) a second epitope tag sequence;

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

(vii) a third epitope tag sequence; and

(viii) a first endoplasmic reticulum (ER) retention sequence,

wherein the second fusion protein comprises, in an N- to C-terminal direction;

(ix) a second endoplasmic reticulum (ER) targeting sequence;

(x) an enzyme, wherein the enzyme is a protease; and

(xi) a second endoplasmic reticulum (ER) retention sequence.

36. The nucleic acid of claim 35, wherein the nucleic acid encoding the first fusion protein is operably linked to a first promoter;

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

37. The nucleic acid of claim 36, wherein the first promoter and the second promoter are expressable in yeast.

38. The nucleic acid of claim 36, wherein the first promoter is Gal1 or Gal10.

39. The nucleic acid of claim 36, wherein the second promoter is Gal1 or Gal10.

40. The nucleic acid of claim 36, wherein the nucleic acid comprises one or more enhancers.

41. The nucleic acid of claim 35, wherein at least a portion of the first peptide is randomized.

42. The nucleic acid of claim 35, wherein the first peptide is the native substrate of the protease.

43. The nucleic acid of claim 35, wherein at least a portion of the second peptide is randomized.

44. The nucleic acid of claim 35, wherein the second peptide is a sequence that is unrelated to the native substrate or shares no or essentially no sequence identity with the native substrate of the protease.

45. The nucleic acid of claim 35, wherein the second peptide is a mutated native substrate of the protease.

46. The nucleic acid of claim 35, wherein the second peptide has 1, 2, 3, 4, or 5 mutations, additions, or deletions as compared to the native substrate of the protease.

47. The nucleic acid of claim 35, wherein the protease is a human protease.

48. The nucleic acid of claim 47 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.

49. The nucleic acid of claim 35, wherein at least a portion of the protease is randomized.

50. The nucleic acid of claim 35, 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.

51. The nucleic acid of claim 35, wherein the first endoplasmic reticulum (ER) targeting sequence and the second endoplasmic reticulum (ER) targeting sequence are MQLLRCFSIFSVIASVLA (SEQ ID NO:

3).

52. The nucleic acid of claim 35, wherein the first endoplasmic reticulum (ER) retention sequence and the second endoplasmic reticulum (ER) retention sequence are FEHDEL (SEQ ID NO:

4), KDEL (SEQ ID NO;

5), HDEL (SEQ ID NO;

6), or RDEL (SEQ ID NO;

7).

53. The nucleic acid of claim 35, wherein the third epitope tag sequence is a hemagglutinin epitope tag.

54. A cell comprising the nucleic acid vector of claim 35.

55. The cell of claim 54, wherein the cell is a yeast cell.

56. A method for producing a protease, comprising:

(i) expressing one or more nucleic acid of claim 35 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.

57. The method of claim 56, wherein the cell is a yeast cell.

58. The method of claim 56, wherein the antibody is labeled with a fluorophore.

59. The method of claim 56, wherein the isolating or purifying the protease comprises FACS.

60. The method of claim 56, further comprising isolating the nucleic acid.

61. The method of claim 60, further comprising further randomizing a portion of the nucleic acid.

62. The method of claim 56, further comprising further characterizing the protease encoded by the nucleic acid.

63. The method of claim 62, further comprising repeating steps (i) and (ii).

64. A nucleic acid vector for engineering kinase variants, wherein the nucleic acid vector encodes two separately expressed fusion proteins, wherein the first fusion protein comprises, in an N- to C-terminal direction:

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

(ii) a surface expression sequence;

(ii) an epitope tag sequence and a peptide sequence that is a selection substrate sequence for the enzyme of the second fusion protein; and

(iii) a first endoplasmic reticulum (ER) retention sequence,

wherein the second fusion protein comprises, in an N- to C-terminal direction;

(iv) a second endoplasmic reticulum (ER) targeting sequence;

(ix) an enzyme, wherein the enzyme is a protein kinase; and

(x) a second endoplasmic reticulum (ER) retention sequence.

65. The nucleic acid of claim 64, wherein the nucleic acid encoding the first fusion protein is operably linked to a first promoter;

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

66. The nucleic acid of claim 65, wherein the first promoter and the second promoter are expressable in yeast.

67. The nucleic acid of claim 65, wherein the first promoter is Gal1 or Gal10.

68. The nucleic acid of claim 65, wherein the second promoter is Gal1 or Gal10.

69. The nucleic acid of claim 65, wherein the nucleic acid comprises one or more enhancers.

70. The nucleic acid of claim 64, wherein at least a portion of the peptide sequence is randomized.

71. The nucleic acid of claim 64, wherein the peptide sequence is the native substrate of the kinase.

72. The nucleic acid of claim 64, wherein the kinase is a tyrosine kinase, an AGC kinase, a CAMK (CaM kinase), a CMGC kinase, a CK1 kinase, a STE kinase, or a TKL kinase, or a thymidine kinase (TK kinase).

73. The nucleic acid of claim 64, wherein the first endoplasmic reticulum (ER) targeting sequence and the second endoplasmic reticulum (ER) targeting sequence are MQLLRCFSIFSVIASVLA (SEQ ID NO:

3).

74. The nucleic acid of claim 64, wherein the first endoplasmic reticulum (ER) retention sequence and the second endoplasmic reticulum (ER) retention sequence are FEHDEL (SEQ ID NO:

4), KDEL (SEQ ID NO;

5), HDEL (SEQ ID NO;

6), or RDEL (SEQ ID NO;

7).

75. A cell comprising the nucleic acid vector of claim 64.

76. The cell of claim 75, wherein the cell is a yeast cell.

77. A method for producing a kinase, comprising:

(i) expressing one or more nucleic acid of claim 64 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 kinase.

78. The method of claim 77, wherein the cell is a yeast cell.

79. The method of claim 77, wherein the antibody is labeled with a fluorophore.

80. The method of claim 77, wherein the isolating or purifying the kinase comprises FACS.

81. The method of claim 77, further comprising isolating the nucleic acid.

82. The method of claim 81, further comprising further randomizing a portion of the nucleic acid.

83. The method of claim 77, further comprising further characterizing the kinase encoded by the nucleic acid.

84. The method of claim 83, further comprising repeating steps (i) and (ii).