Protein Surface Remodeling

• US 20100209994A1
• Filed: 06/01/2007
• Published: 08/19/2010
• Est. Priority Date: 06/02/2006
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Lawrence, Michael S., Phillips, Kevin John, Liu, David R.
• Granted Patent: US 9,150,626 B2
• Time in Patent Office: Days
• US Class Current: 435/193
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12Y 603/02003 Glutathione synthase (6.3.2.3)

Abstract

Aggregation is a major cause of the misbehavior of proteins. A system for modifying a protein to create a more stable variant is provided. The method involves identifying non-conserved hydrophobic amino acid residues on the surface of a protein, suitable for mutating to more hydrophilic residues (e.g., charged amino acids). Any number of residues on the surface may be changed to create a variant that is more soluble, resistant to aggregation, has a greater ability to re-fold, and/or is more stable under a variety of conditions. The invention also provides GFP, streptavidin, and GST variants with an increased theoretical net charge created by the inventive technology. Kits are also provided for carrying out such modifications on any protein of interest.

Claims

1. A method of improving the stability of a protein of interest, the method comprising steps of:

identifying the surface residues of a protein of interest that are not highly conserved among other proteins related to the protein of interest; and

replacing a plurality of non-conserved, surface residue with an amino acid residue that is positively charged at physiological pH.

3. The method of claim 1, wherein the non-conserved, surface residues are hydrophobic.

4. The method of claim 1, wherein the non-conserved, surface residues are hydrophilic or negatively charged.

2. A method of improving the stability of a protein of interest, the method comprising steps of:

identifying the surface residues of a protein of interest that are not highly conserved among other proteins related to the protein of interest; and

replacing a plurality of non-conserved, surface residue with an amino acid residue that is negatively charged at physiological pH.

5. (canceled)

6. A method of improving the stability of a protein of interest, the method comprising steps of:

identifying surface residues of a protein of interest;

identifying the surface residues of the protein of interest that are not highly conserved among other proteins related to the protein of interest;

assigning a hydrophobicity value to each of the identified non-conserved surface residues; and

replacing at least one surface residue with an amino acid residue that is charged at physiological pH, wherein the residues being replaced are all mutated into the same type of residue, wherein the type of residue is either positively charged residues or negatively charged residues.

7. The method of claim 6, wherein the step of replacing comprises replacing at least one hydrophobic surface residue.

8. The method of claim 6, wherein the step of replacing comprises replacing at least one hydrophilic surface residue.

9. The method of claim 6, wherein the step of replacing comprises replacing at least one charged surface residue.

10. The method of claim 6, wherein the step of replacing comprises replacing at least one surface residue with a lysine residue.

11. The method of claim 6, wherein the step of replacing comprises replacing at least one surface residue with an aspartate or glutamate residue.

12. The method of claim 6, wherein the step of replacing comprises replacing at least two surface residues.

13. The method of claim 6, wherein the step of replacing comprises replacing at least five surface residues.

14. The method of claim 6, wherein the step of replacing comprises replacing at least ten surface residues.

15. The method of claim 6, wherein the step of replacing comprises replacing at least twenty surface residues.

16. The method of claim 6, wherein the step of replacing comprises replacing at least thirty surface residues.

17. The method of claim 6, whereby the method creates a modified protein with a greater net charge at physiological pH than the original protein of interest.

18. The method of claim 6, whereby the method creates a modified protein of interest that is more negatively charged at physiological pH than the original protein of interest.

19. The method of claim 6, whereby the method creates a modified protein of interest that is at least −

5 more negatively charged at physiological pH than the original protein of interest.

20. The method of claim 6, whereby the method creates a modified protein of interest that is at least −

10 more negatively charged at physiological pH than the original protein of interest.

21. The method of claim 6, whereby the method creates a modified protein of interest that is at least −

15 more negatively charged at physiological pH than the original protein of interest.

22. The method of claim 6, whereby the method creates a modified protein of interest that is at least −

20 more negatively charged at physiological pH than the original protein of interest.

23. The method of claim 6, whereby the method creates a modified protein of interest that is more positively charged at physiological pH than the original protein of interest.

24. The method of claim 6, whereby the method creates a modified protein of interest that is at least +5 more positively charged at physiological pH than the original protein of interest.

25. The method of claim 6, whereby the method creates a modified protein of interest that is at least +10 more positively charged at physiological pH than the original protein of interest.

26. The method of claim 6, whereby the method creates a modified protein of interest that is at least +15 more positively charged at physiological pH than the original protein of interest.

27. The method of claim 6, whereby the method creates a modified protein of interest that is at least +20 more positively charged at physiological pH than the original protein of interest.

28. The method of claim 6, wherein the protein of interest is a protein susceptible to aggregation.

29. The method of claim 6, wherein the protein of interest is a hydrophobic protein.

30. The method of claim 6, wherein the protein of interest is a membrane protein.

31. The method of claim 6, wherein the protein of interest is a protein that is difficult to overexpress.

32. The method of claim 6, wherein the protein of interest is a protein that is difficult to purify.

33. The method of claim 6, wherein the protein of interest is a receptor.

34. The method of claim 6, wherein the protein of interest is a transcription factor.

35. The method of claim 6, wherein the protein of interest is an enzyme.

36. The method of claim 6, wherein the protein of interest is a structural protein.

37. The method of claim 6, wherein the protein of interest is a fluorescent protein.

38. The method of claim 6, wherein the protein of interest is green fluorescent protein (GFP).

39. The method of claim 6, wherein the protein of interest is an extracellular protein.

40. The method of claim 6, wherein the protein of interest is streptavidin.

41. The method of claim 6, wherein the protein of interest is glutathione-S-transferase.

42. The method of claim 6, wherein the step of identifying the surface residues comprises computer modeling the three-dimensional structure of the protein.

43. The method of claim 6, wherein the step of identifying the surface residues comprises predicting using algorithms whether a residue is found on the surface of a protein.

44. The method of claim 6, wherein the step of identifying the surface residues comprises identifying residues with an avNAPSA value less than a threshold value.

45. The method of claim 6, wherein the step of identifying the surface residues that are not highly conserved comprises aligning the amino acid sequence of the protein with at least one other protein from the same protein family.

46. The method of claim 6, wherein the step of identifying the surface residues that are not highly conserved comprises aligning the amino acid sequence of the protein with at least one other amino acid sequence of the protein from a different species.

47. The method of claim 45, wherein the alignment is performed with at least 2 other protein sequences.

48. The method of claim 45, wherein the alignment is performed with at least 3 other protein sequences.

49. The method of claim 45, wherein the alignment is performed with at least 5 other protein sequences.

50. The method of claim 6, wherein the step of assigning a hydrophobicity value comprises using octanol/water P values.

51. The method of claim 6, wherein the step of replacing comprises mutagenizing the sequence of the protein to replace the identified hydrophobic surface residue with a natural amino acid residue that is charged at physiological pH.

52. The method of claim 51, wherein the natural amino acid residue is selected from the group consisting of lysine, glutamate, aspartate, histidine, and arginine.

53. The method of claim 51, wherein the natural amino acid residue is selected from the group consisting of lysine, histidine, and arginine.

54. The method of claim 51, wherein the natural amino acid residue is lysine.

55. The method of claim 51, wherein the natural amino acid residue is selected from the group consisting of glutamate and aspartate.

56. The method of claim 6, wherein the step of replacing comprises site-directed mutagenesis of the identified surface residues.

57. The method of claim 6, wherein the step of replacing comprises PCR mutagenesis of the identified surface residues.

58. A green fluorescent protein (+36 GFP) of amino acid sequence:

59. A polynucleotide encoding the green fluorescent protein of claim 58.

60. The polynucleotide of claim 59 of sequence:

61. A green fluorescent protein (+49 GFP) of amino acid sequence:

62. A polynucleotide encoding the green fluorescent protein of claim 61.

63. The polynucleotide of claim 62 of sequence:

64-78. -78. (canceled)