Methods and Compositions Related to Cyclic Peptide Synthesis

1 . An isolated peptide comprising an amino acid segment comprising the amino acid sequence of SEQ ID NO: 1, an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO:1, or the amino acid sequence of SEQ ID NO:1 having one or more conservative amino acid substitutions.

2 . The isolated peptide of claim 1 , wherein “N 1 ” and “N 2 ” of SEQ ID NO: 1 represent coding sequences.

3 . The isolated peptide of claim 2 , wherein the coding sequences have a length of less than 100 residues.

4 . The isolated peptide of claim 2 , wherein the coding sequences have a length of less than 50 residues.

5 . The isolated peptide of claim 2 , wherein the coding sequences have a length of less than 20 residues.

6 . The isolated peptide of claim 2 , wherein the coding sequences have a length of less than 10 residues.

7 . An isolated peptide comprising an amino acid segment comprising the amino acid sequence of SEQ ID NO: 50, an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO:50, or the amino acid sequence of SEQ ID NO:50 having one or more conservative amino acid substitutions.

8 . The isolated peptide of claim 7 , wherein “N 1 ” and “N 2 ” of SEQ ID NO: 50 represent coding sequences.

9 . The isolated peptide of claim 8 , wherein the coding sequences have a length of less than 100 residues.

10 . The isolated peptide of claim 8 , wherein the coding sequences have a length of less than 50 residues.

11 . The isolated peptide of claim 8 , wherein the coding sequences have a length of less than 20 residues.

12 . The isolated peptide of claim 8 , wherein the coding sequences have a length of less than 10 residues.

13 . An isolated peptide comprising an amino acid segment comprising the amino acid sequence of SEQ ID NO: 2, an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 2, or the amino acid sequence of SEQ ID NO: 2 having one or more conservative amino acid substitutions.

14 . An isolated peptide comprising an amino acid segment comprising the amino acid sequence of SEQ ID NO: 3, an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence of SEQ ID NO: 3 having one or more conservative amino acid substitutions.

15 . An isolated peptide comprising an amino acid segment comprising the amino acid sequence of SEQ ID NO: 4, an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 4, or the amino acid sequence of SEQ ID NO: 4 having one or more conservative amino acid substitutions.

16 . An isolated peptide comprising an amino acid segment comprising the amino acid sequence of SEQ ID NO: 5, an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence of SEQ ID NO: 5 having one or more conservative amino acid substitutions.

17 . An isolated peptide comprising an amino acid segment comprising the amino acid sequence of SEQ ID NO: 6, an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 6, or the amino acid sequence of SEQ ID NO: 6 having one or more conservative amino acid substitutions.

18 . An isolated peptide comprising an amino acid segment comprising the amino acid sequence of SEQ ID NO: 7, an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 7, or the amino acid sequence of SEQ ID NO: 7 having one or more conservative amino acid substitutions.

19 . An isolated peptide comprising an amino acid segment comprising the amino acid sequence of SEQ ID NO: 8, an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 8, or the amino acid sequence of SEQ ID NO: 8 having one or more conservative amino acid substitutions.

20 . An isolated peptide comprising an amino acid segment comprising the amino acid sequence of SEQ ID NO: 9, an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 9, or the amino acid sequence of SEQ ID NO: 9 having one or more conservative amino acid substitutions.

21 . The isolated peptide of claim 20 , wherein N of SEQ ID NO: 9 represents a coding sequence.

22 . The isolated peptide of claim 21 , wherein the coding sequence has a length of less than 100 residues.

23 . The isolated peptide of claim 21 , wherein the coding sequence has a length of less than 50 residues.

24 . The isolated peptide of claim 21 , wherein the coding sequence has a length of less than 20 residues.

25 . The isolated peptide of claim 21 , wherein the coding sequence has a length of less than 10 residues.

26 . A vector comprising a nucleotide sequence encoding a fusion polypeptide comprising, from N-terminus to C-terminus: a) a C-terminal domain comprising SEQ ID NO: 10; b) a peptide; c) an N-terminal domain comprising SEQ ID NO: 11; wherein the fusion polypeptide is able to cyclize the peptide to produce a cyclic peptide in a mammalian cell.

27 . A vector comprising a nucleotide sequence encoding a fusion polypeptide comprising, from N-terminus to C-terminus: a) a C-terminal domain comprising SEQ ID NO: 11; b) a peptide; c) an N-terminal domain comprising SEQ ID NO: 12; wherein the fusion polypeptide is able to cyclize the peptide to produce a cyclic peptide in a mammalian cell.

28 . A vector comprising a nucleotide sequence encoding a fusion polypeptide comprising, from N-terminus to C-terminus: a) a C-terminal domain comprising SEQ ID NO: 10; b) a peptide; c) an N-terminal domain comprising SEQ ID NO: 12; wherein the fusion polypeptide is able to cyclize the peptide to produce a cyclic peptide in a mammalian cell.

29 . A vector comprising a nucleotide sequence encoding a fusion polypeptide comprising, from N-terminus to C-terminus: a) a C-terminal domain comprising SEQ ID NO: 13; b) a peptide; c) an N-terminal domain comprising SEQ ID NO: 14; wherein the fusion polypeptide is able to cyclize the peptide to produce a cyclic peptide in a mammalian cell.

30 . The vector of any one of claims 26 - 29 in which the peptide is a random peptide.

31 . The vector of any one of claims 26 - 29 in which the peptide of interest is derived from a cDNA library.

32 . A library of vectors of any one of claims 26 - 29 in which each vector in the library encodes a different fusion polypeptide.

33 . The library of claim 32 in which the peptide of interest of each different fusion polypeptide is different.

34 . The library of claim 32 in which the peptide of interest is a random peptide at least 3 amino acids in length.

35 . A cell comprising the vector of any one of claims 26 - 29 , or progeny thereof.

36 . The cell of claim 35 which is a prokaryotic cell.

37 . The cell of claim 35 which is a eukaryotic cell.

38 . The cell of claim 37 which is a mammalian cell.

39 . The cell of claim 38 which is selected from the group consisting of a tumor cell, a liver cell, a hepatocyte, a mast cell and a lymphocyte cell.

40 . The cell of claim 38 which is a human cell.

41 . A method for cyclizing a polypeptide, the method comprising inserting the polypeptide to be cyclized in the “N 1 ” or “N 2 ” position of SEQ ID NO: 1.

42 . A method for cyclizing a polypeptide, the method comprising inserting the polypeptide to be cyclized in the “N” position of SEQ ID NO: 2.

43 . A method for cyclizing a polypeptide, the method comprising inserting the polypeptide to be cyclized in the “N” position of SEQ ID NO: 3.

44 . A method for cyclizing a polypeptide, the method comprising inserting the polypeptide to be cyclized in the “N” position of SEQ ID NO: 9.

45 . A method for cyclizing a polypeptide, the method comprising expressing the retroviral vector of any one of claims 26 - 31 .

46 . A method for cyclizing a polypeptide, the method comprising inserting the polypeptide to be cyclized in the “N 1 ” or “N 2 ” position of SEQ ID NO: 50.

47 . A method for prenylating a polypeptide, the method comprising inserting the polypeptide to be cyclized in the “N 1 ” or “N 2 ” position of SEQ ID NO: 50.

48 . A method for screening for a cyclized peptide that interacts with a given composition, the method comprising exposing the given composition to a cyclized peptide, and determining interaction between the cyclized peptide and the given composition.

49 . The method of claim 48 , wherein the cyclized peptide is part of a library of cyclized peptides.

50 . The method of claim 48 , wherein the given composition is a cell, protein, peptide, antibody, small molecule, or nucleic acid.

51 . The method of claim 48 , wherein the given composition is associated with a disease or disorder.

52 . The method of claim 51 , wherein the disease or disorder is cancer or an infectious disease.

53 . The method of claim 48 , wherein the interaction between the cyclized peptide and the given composition is determined by array, microscopy, phage display, or yeast two-hybrid system.

54 . The method of claim 48 , wherein the cyclized peptide is produced by the method of claim 41 .

55 . The method of claim 48 , wherein the cyclized peptide is produced by the method of claim 42 .

56 . The method of claim 48 , wherein the cyclized peptide is produced by the method of claim 43 .

57 . The method of claim 48 , wherein the cyclized peptide is produced by the method of claim 44 .

58 . The method of claim 48 , wherein the cyclized peptide is produced by the method of claim 45 .

59 . The method of claim 48 , wherein the cyclized peptide is produced by the method of claim 46 .

60 . A cyclized peptide identified by the method of claim 48 .

61 . An array comprising a library of cyclized peptides.

62 . The array of claim 61 , wherein the cyclized peptides are produced by the method of claim 41 .

63 . The array of claim 61 , wherein the cyclized peptides are produced by the method of claim 42 .

64 . The array of claim 61 , wherein the cyclized peptides are produced by the method of claim 43 .

65 . The array of claim 61 , wherein the cyclized peptides are produced by the method of claim 44 .

66 . The array of claim 61 , wherein the cyclized peptides are produced by the method of claim 45 .

67 . The array of claim 61 , wherein the cyclized peptides are produced by the method of claim 46 .

68 . An isolated peptide comprising an amino acid segment comprising the amino acid sequence of SEQ ID NO: 52, an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 52, or the amino acid sequence of SEQ ID NO: 52 having one or more conservative amino acid substitutions.

69 . An isolated peptide comprising an amino acid segment comprising the amino acid sequence of SEQ ID NO: 53, an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 53, or the amino acid sequence of SEQ ID NO: 53 having one or more conservative amino acid substitutions.

70 . A vector comprising a nucleotide sequence encoding a fusion polypeptide comprising, from N-terminus to C-terminus: a) a C-terminal domain comprising recognition sequence; b) a peptide; c) an N-terminal domain comprising a recognition sequence; wherein the fusion polypeptide is able to cyclize the peptide to produce a cyclic peptide in a mammalian cell.

71 . A vector comprising a nucleotide sequence encoding a fusion polypeptide comprising, from N-terminus to C-terminus: a) a C-terminal domain comprising the motif GXXXS (SEQ ID NO: 35); b) a peptide; c) an N-terminal domain comprising the motif XYDG (SEQ ID NO: 36); wherein the fusion polypeptide is able to cyclize the peptide to produce a cyclic peptide in a mammalian cell.

72 . The vector of claim 71 , wherein the C-terminal domain comprises SEQ ID NO: 17.

73 . The vector of claim 72 , wherein the N-terminal domain comprises SEQ ID NO: 18.

74 . The vector of claim 71 , wherein the N-terminal domain comprises SEQ ID NO: 18.

75 . A method of making fusion polypeptide comprising fusing one or more peptides into a vector comprising two or more recognition sequences; wherein the one or more peptides are inserted between the two or more recognition sequences; and wherein the one or more peptides is cyclized.

76 . The method of claim 75 , wherein the two or more recognition sequences are selected from the group consisting of SEQ ID NO: 10 and 11; SEQ ID NO: 11 and 12; SEQ ID NO: 10 and 12; SEQ ID NO: 13 and 14; and SEQ ID NO: 17 and 18.

77 . A method of screening for bioactive compounds comprising constructing a vector comprising, from N-terminus to C-terminus: a) a C-terminal recognition sequence; b) a peptide; and c) a N-terminal recognition sequence; wherein the polypeptide is able to cyclize the peptide; creating a library of one or more cyclized peptides; contacting a cell, protein, peptide, or nucleic acid with the one or more cyclized peptides; monitoring the cell, protein, peptide, or nucleic acid for the desired response; wherein the presence of the desired response indicates a bioactive compound.

78 . The method of claim 77 , wherein the two or more recognition sequences are selected from the group consisting of SEQ ID NO: 10 and 11; SEQ ID NO: 11 and 12; SEQ ID NO: 10 and 12; SEQ ID NO: 13 and 14; and SEQ ID NO: 17 and 18.

79 . The method of claim 77 , wherein the one or more cyclic peptides are introduced into a cell.

80 . The method of claim 77 , wherein the cell, protein, peptide, or nucleic acid is contacted with the cyclized peptide in an expression array.

81 . The method of claim 77 , wherein the cell is monitored by array, microscopy, phage display, or yeast two-hybrid system.

82 . A method of creating a cyclic peptide library comprising cloning DNA from a cyanobacteria source into a vector; transforming bacteria with the vector containing the source DNA; ligating into the vector a nucleic acid encoding a peptide; wherein the peptide is inserted in the coding region of the source DNA and is flanked by two or more recognition sequences.

83 . The method of claim 82 , wherein the recognition sequences are selected from the group consisting of SEQ ID NO: 10 and 11; SEQ ID NO: 11 and 12; SEQ ID NO: 10 and 12; SEQ ID NO: 13 and 14; and SEQ ID NO: 17 and 18.

84 . A method of making a cyclic peptide comprising creating a cyclic peptide library according to claim 82 and inducing expression of a peptide encoded by the nucleic acid and purifying the recombinant peptide.