Methods and Compositions Related to Cyclic Peptide Synthesis

Abstract

Disclosed are compositions and methods for cyclization of polymers such as peptides.

What is claimed is: 
     
         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.