KINETICALLY EFFICIENT SUBSTRATE FOR LIPOIC ACID LIGASE

• US 20110130348A1
• Filed: 10/19/2010
• Published: 06/02/2011
• Est. Priority Date: 10/19/2009
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: PUTHENVEETIL, SUJIET, TING, ALICE Y.
• Granted Patent: US 8,759,482 B2
• Time in Patent Office: Days
• US Class Current: 514/21.5
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68

Abstract

The invention provides methods for identifying and optimizing peptide substrates for enzymes such as lipoic acid ligase (Lp1A).

Claims

1. A lipoic acid ligase (Lp1A) acceptor peptide, wherein the peptide comprises 8-13 amino acids, including a central lysine residue at position 0, a valine residue at position +1, a tryptophan residue at position +2, a glutamic acid or aspartic acid residue at position +4, a hydrophobic residue at position +5, a glutamic acid residue at position −

3, and a phenylalanine residue at position −

4.

3. The peptide of claim 1, wherein the peptide comprises the sequence GFEIDKVWYDLDA (SEQ ID NO:

1).

4. The peptide of claim 3, wherein the peptide consists of the sequence GFEIDKVWYDLDA (SEQ ID NO:

1).

5. A nucleic acid encoding the peptide of claim 1.

6. A composition comprising the peptide of claim 1 and a carrier.

7. A composition comprising the peptide of claim 1 wherein the peptide is N- or C-terminally fused to a target protein, and a carrier.

2. (canceled)

2-1. -29. (canceled)

8. A lipoic acid ligase (Lp1A) acceptor peptide, wherein the peptide comprises 8-13 amino acids, including a central lysine residue at position 0, a hydrophobic residue at position +1, an aromatic residue at position +2, an aromatic or aliphatic hydrophobic residue at position +3, a glutamic acid or aspartic acid residue at position +4, an aliphatic hydrophobic residue at position +5, an aspartic acid, asparagine, glutamic acid, tyrosine or alanine residue at position −

1, a glutamic acid or aspartic acid residue at position −

3, and a hydrophobic or aromatic residue at position −

4.

9. The peptide of claim 8, wherein position +7 is a serine residue, an alanine residue, or is absent.

10. The peptide of claim 8, wherein position −

5 is a glycine residue or is absent.

12. The peptide of claim 8, wherein the residue at position +1 is a valine, isoleukine, leucine, or phenylalanine residue.

16. The peptide of claim 8, wherein the residue at position +2 is a tryptophan or phenylalanine residue.

18. The peptide of claim 8, wherein the residue at position +3 is a tyrosine, histidine, phenylalanine, valine, leucine, threonine, or an isoleucine residue.

25. The peptide of claim 8, wherein the residue at position +4 is a glutamic acid or an aspartic acid residue.

27. The peptide of claim 8, wherein the residue at position +5 is a leucine, phenylalanine, or an isoleucine residue.

30. The peptide of claim 8, wherein the residue at position +6 is an aspartic acid, a glutamic acid, serine, threonine, cysteine, or tyrosine residue.

36. The peptide of claim 8, wherein the residue at position −

1 is an aspartic acid, asparagine, alanine, a glutamic acid, or tyrosine residue.

41. The peptide of claim 8, wherein the residue at position −

2 is an isoleucine, arginine, a histidine, or leucine residue.

45. The peptide of claim 8, wherein the residue at position −

3 is a glutamic acid or an aspartic acid residue.

47. The peptide of claim 8, wherein the residue at position −

4 is a phenylalanine, valine, leucine, or an isoleucine residue.

51. The peptide of claim 8, wherein the peptide comprises the sequence GFEIDKVWYDLDA (SEQ ID NO:

1).

52. The peptide of claim 51, wherein the peptide consists of the sequence GFEIDKVWYDLDA (SEQ ID NO:

1).

53. A nucleic acid encoding the peptide of claim 8.

54. A composition comprising the peptide of claim 8 or the nucleic acid of claim 53, and a carrier.

55. A composition comprising the peptide of claim 8, wherein the peptide is N- or C-terminally fused to a target protein, and a carrier.

11. (canceled)

13-15. -15. (canceled)

17. (canceled)

19-24. -24. (canceled)

26. (canceled)

31-35. -35. (canceled)

37-40. -40. (canceled)

42-44. -44. (canceled)

46. (canceled)

48-50. -50. (canceled)

56. A method for identifying an acceptor peptide that functions as a substrate for an enzyme, for use in protein labeling, the method comprising:

performing surface display in cells, wherein each cell expresses one acceptor peptide that is fused to a cell surface protein,

labeling each cell with the enzyme to ligate the acceptor peptide to a probe,

sorting each cell based on the extent of acceptor peptide ligation, and

selecting an acceptor peptide that has a k_cat between 0.001 s<sup>−

1</sup>-1 s<sup>−

1</sup> and a K_m between 500 μ

M-1 μ

M,

wherein an acceptor peptide that has a k_cat between 0.001 s<sup>−

1</sup>-1 s<sup>−

1</sup>and a K_m between 500 μ

M-1 μ

M is an acceptor peptide that functions as a substrate for the enzyme for use in protein labeling.

57-64. -64. (canceled)