Protein surface remodeling

• US 9,434,774 B2
• Filed: 12/30/2011
• Issued: 09/06/2016
• 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: Liu, David R., Phillips, Kevin John, Lawrence, Michael S.
• Primary Examiner(s): RIGGS II, LARRY D
• Application Number:
  US13/341,231
• Publication Number:
  US 20120129759A1
• Time in Patent Office: 1,712 Days
• Field of Search: None
• US Class Current: 1/1
• 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 supercharged protein variant of a wild-type protein, wherein the supercharged protein variant comprises a modified primary amino acid sequence as compared to the wild-type sequence, resulting in a theoretical net charge on the supercharged protein variant of +10 to +52 at physiological pH, wherein the theoretical net charge at physiological pH of the supercharged protein variant is increased by at least +3 as compared to the theoretical net charge of the wild-type protein,

wherein the wild-type protein is an immunoglobulin or a fragment thereof.

2. The supercharged protein of claim 1, wherein the theoretical net charge at physiological pH of the supercharged protein variant is increased by at least +4, at least +5, at least +10, at least +15, at least +20, at least +25, at least +30, or at least +35 as compared to the theoretical net charge of the wild-type sequence.

3. The supercharged protein variant of claim 1, wherein the theoretical net charge at physiological pH of the supercharged protein is within the range of +52 to +20, +52 to +30, or +52 to +40.

4. The supercharged protein variant of claim 1, wherein the supercharged protein variant retains at least 50%, at least 75%, at least 90%, or at least 95% of the activity of the wild-type protein.

5. The supercharged protein variant of claim 1, wherein the wild-type protein is an immunoglobulin.

6. The supercharged protein variant of claim 1, wherein the immunoglobulin or fragment thereof is human or humanized.

7. The supercharged protein variant of claim 1, wherein the variant is a fusion protein.

8. The supercharged protein variant of claim 7, wherein the fusion protein comprises a linker.

9. The supercharged protein variant of claim 1, wherein the modified primary amino acid sequence of the supercharged protein variant comprises a replacement of at least one charged surface residue of the wild-type protein with a different residue.

10. The supercharged protein variant of claim 1, wherein the modified primary amino acid sequence of the supercharged protein variant comprises a replacement of at least one surface residue of the wild-type protein with a lysine, histidine, or arginine residue.

11. The supercharged protein variant of claim 1, wherein the modified primary amino acid sequence of the supercharged protein variant comprises a replacement of at least two, at least five, at least ten, at least twenty, or at least thirty surface residues of the wild-type protein with a different residue.

12. A composition comprising the supercharged protein variant of claim 1.

13. The supercharged protein variant of claim 1, wherein the wild-type protein is a fragment of an immunoglobulin.

14. The supercharged protein variant of claim 1,

wherein the modified primary amino acid sequence comprises replacing a plurality of non-conserved, surface residues with a natural amino acid residue that is positively charged at physiological pH;

and

wherein non-conserved, surface residues are identified by comparing the amino acid sequence of the protein with at least one other amino acid sequence of the protein from the same protein family or a different species, wherein a residue is non-conserved if less than or equal to 50% of the amino acid sequences have the same amino acid sequence in a particular position.

15. A method of preparing a supercharged protein variant of claim 1, 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 residues with an amino acid residue that is positively charged at physiological pH, or

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

16. A supercharged protein variant of a wild-type protein, wherein the supercharged protein variant comprises a modified primary amino acid sequence as compared to the wild-type sequence, resulting in a theoretical net charge on the supercharged protein variant of −

40 to −

10 at physiological pH, wherein the theoretical net charge at physiological pH of the supercharged protein variant is decreased by at least −

3 as compared to the theoretical net charge of the wild-type protein,

wherein the wild-type protein is an immunoglobulin or a fragment thereof.

17. The supercharged protein of claim 16, wherein the theoretical net charge at physiological pH of the supercharged protein variant is decreased by at least −

4, at least −

5, at least −

10, at least −

15, at least −

20, at least −

25, at least −

30, or at least −

35 as compared to the theoretical net charge of the wild-type sequence.

18. The supercharged protein variant of claim 16, wherein the theoretical net charge at physiological pH of the supercharged protein is within the range of −

40 to −

20 or −

40 to −

30.

19. The supercharged protein variant of claim 16, wherein the modified primary amino acid sequence of the supercharged protein variant comprises a replacement of at least one charged surface residue of the wild-type protein with a different residue.

20. The supercharged protein variant of claim 16, wherein the modified primary amino acid sequence of the supercharged protein variant comprises a replacement of at least one surface residue of the wild-type protein with an aspartate or glutamate residue.

21. The supercharged protein variant of claim 16, wherein the wild-type protein is an immunoglobulin.

22. The supercharged protein variant of claim 16, wherein the wild-type protein is a fragment of an immunoglobulin.

23. The supercharged protein variant of claim 16, wherein the immunoglobulin or fragment thereof is human or humanized.

24. The supercharged protein variant of claim 16,

wherein the modified primary amino acid sequence comprises replacing a plurality of non-conserved, surface residues with a natural amino acid residue that is positively charged at physiological pH;

and

wherein non-conserved, surface residues are identified by comparing the amino acid sequence of the protein with at least one other amino acid sequence of the protein from the same protein family or a different species, wherein a residue is non-conserved if less than or equal to 50% of the amino acid sequences have the same amino acid sequence in a particular position.

25. A protein selected from:

a green fluorescent protein (+36 GFP) comprising the amino acid sequence;

MGHHHHHHGGASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFICTT GKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRA EVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNV KDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKH GRDERYK (SEQ ID NO;

     5);

a green fluorescent protein (+42 GFP) comprising the amino acid sequence;

MGHHHHHHGGRSKGKRLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFICT TGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTR AEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHN VKDGSVQLADHYQQNTPIGRGPVLLPRKHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIK HGRKERYK (SEQ ID NO;

     6);

a green fluorescent protein (+48 GFP) comprising the amino acid sequence;

MGHHHHHHGGRSKGKRLFRGKVPILVKLKGDVNGHKFSVRGKGKGDATRGKLTLKFICT TGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTR AEVKFKGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHN VKDGSVQLAKHYQQNTPIGRGPVLLPRKHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIK HGRKERYK (SEQ ID NO;

     27);

a green fluorescent protein (+49 GFP) comprising the amino acid sequence;

MGHHHHHHGGRSKGKRLFRGKVPILVKLKGDVNGHKFSVRGKGKGDATRGKLTLKFICT TGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTR AEVKFKGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHN VKDGSVQLAKHYQQNTPIGRGPVLLPRKHYLSTRSKLSKDPKEKRDHMVLKEFVTAAGIK HGRKERYK (SEQ ID NO;

     7);

a streptavidin (+52 SAV) comprising an amino acid sequence;

MGHHHHHHGGAKAGITGTWYNQLGSTFIVTAGAKGALTGTYESAVGNAKSRYVLTGRYD SAPATKGSGTALGWTVAWKNKYRNAHSATTWSGQYVGGAKARINTQWLLTSGTTKAKA WKSTLVGHDTFTKVKPSAAS (SEQ ID NO;

     30); and

a glutathione-S-transferase (+50 GST) of amino acid sequence;

MGHHHHHHGGPPYTITYFPVRGRCEAMRMLLADQKQSWKEEVVTMKTWPPLKPSCLFRQ LPKFQDGKLTLYQSNAILRHLGRSFGLYGKKQKEAALVDMVNDGVEDLRCKYATLIYTKY KAGKKKYVKKLPKHLKPFETLLSKNKGGKAFVVGSKISFADYNLLDLLRIHQVLNPSCLKA FPLLSAYVARLSARPKIKAFLASPEHVKRPINGNGKQ (SEQ ID NO;

     35).

26. A supercharged protein selected from:

a streptavidin protein (−

40 SAV) comprising an amino acid sequence;

MGHHHHHHGGAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGDAESEYVLTGRYD SAPATDGSGTALGWTVAWKNDYENAHSATTWSGQYVGGAEARINTQWLLTSGTTEADA WKSTLVGHDTFTKVEPSAAS (SEQ ID NO;

     29);

a green fluorescent protein (−

30 GFP) of amino acid sequence;

MGHHHHHHGGASKGEELFDGVVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKFICTT GELPVPWPTLVTTLTYGVQCFSDYPDHMDQHDFFKSAMPEGYVQERTISFKDDGTYKTRA EVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAEFEIRHNVE DGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHMVLLEFVTAAGIDHG MDELYK (SEQ ID NO;

     4);

a green fluorescent protein (−

29 GFP) of amino acid sequence;

MGHHHHHHGGASKGEELFDGEVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKFICTT GELPVPWPTLVTTLTYGVQCFSRYPDHMDQHDFFKSAMPEGYVQERTISFKDDGTYKTRA EVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAEFEIRHNVE DGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHMVLLEFVTAAGIDHG MDELYK (SEQ ID NO;

     3);

a green fluorescent protein (−

25 GFP) of amino acid sequence;

MGHHHHHHGGASKGEELFTGVVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKFICTT GELPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRA EVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAEFEIRHNVE DGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHMVLLEFVTAAGIDHG MDELYK (SEQ ID NO;

     2); and

a glutathione-S-transferase (−

40 GST) of amino acid sequence;

MGHHHHHHGGPPYTITYFPVRGRCEAMRMLLADQDQSWEEEVVTMETWPPLKPSCLFRQ LPKFQDGDLTLYQSNAILRHLGRSFGLYGEDEEEAALVDMVNDGVEDLRCKYATLIYTDY EAGKEEYVEELPEHLKPFETLLSENEGGEAFVVGSEISFADYNLLDLLRIHQVLNPSCLDAFP LLSAYVARLSARPEIEAFLASPEHVDRPINGNGKQ (SEQ ID NO;

     34).