Modified peptide display

Abstract

The invention provides a replicable genetic package displaying a cyclic peptide having at least one intramolecular bond between amino acid side chains. Also provided are a method of preparing such a genetic package displaying cyclic peptides having at least one intramolecular bond. Further provided is a library of replicable genetic packages displaying cyclic peptides each having at least one intramolecular cyclic bond between amino acid side chains; and a method of producing such a library.

What is claimed is: 
     
         1 . A replicable genetic package displaying a peptide having a plurality of amino acid side chains and at least one intramolecular cyclic bond between two of the amino acid side chains. 
     
     
         2 . The replicable genetic package of  claim 1 , wherein the replicable genetic package is selected from the group consisting of a phage particle, a bacterium, a yeast, a fungus, a spore of a microorganism and a ribosome. 
     
     
         3 . The replicable genetic package of  claim 1 , wherein the replicable genetic package is selected from the group consisting of M13, T4, T7, fd and lambda phages. 
     
     
         4 . The replicable genetic package of  claim 1 , wherein the peptide is a precursor peptide comprising a leader and a core peptide. 
     
     
         5 . The replicable genetic package of  claim 4 , wherein the core peptide is derived from a natural ribosomal peptide. 
     
     
         6 . The replicable genetic package of  claim 1 , wherein the cyclic bond links two atoms independently selected from the group consisting of C, N, O and S. 
     
     
         7 . The replicable genetic package of  claim 6 , wherein the cyclic bond is between C—N, C—O, C—S, N—N, N—O, N—S, O—O, or O—S. 
     
     
         8 . The replicable genetic package of  claim 1 , wherein the peptide comprises a polycyclic structure. 
     
     
         9 . The replicable genetic package of  claim 1 , wherein the peptide comprises a randomized amino acid sequence. 
     
     
         10 . A replicable genetic package displaying a peptide having a plurality of amino acid side chains and at least one intramolecular cyclic bond between two atoms of the amino acid side chains, wherein the cyclic bond is between C—N, C—O, C—S, N—N, N—O, N—S, O—O, or O—S, and wherein the replicable genetic package is produced by a method comprising the following steps:
 providing a nucleic acid sequence encoding a peptide of interest; 
 ligating the nucleic acid sequence into a gene of a replicable genetic package; 
 displaying a corresponding primary peptide on a surface of the replicable genetic package to obtain a primary replicable genetic package; and 
 enzymatically processing the primary replicable genetic package with a post-translationally modifying (PTM) enzyme to produce a mature replicable genetic package displaying a cyclic or polycyclic structure of the primary peptide. 
 
     
     
         11 . The replicable genetic package of  claim 10 , wherein the replicable genetic package is selected from the group consisting of a phage particle, a bacterium, a yeast, and a fungus. 
     
     
         12 . The replicable genetic package of  claim 11 , wherein the phage particle is a bacteriophage selected from the group consisting of M13 phage, T4 phage, T7 phage, fd phage, and lambda phage. 
     
     
         13 . The replicable genetic package of  claim 10 , wherein the primary peptide comprises a polycyclic structure. 
     
     
         14 . A method of producing a library of replicable genetic packages displaying a peptide having at least one intramolecular cyclic bond between amino acid side chains, comprising:
 a) providing a repertoire of nucleic acid sequences encoding peptide variants;   b) ligating the repertoire into the genes of replicable genetic packages;   c) displaying the corresponding primary peptide sequences on the surface of the packages to obtain a primary library; and   d) enzymatically processing the primary library with a post-translationally modifying (PTM) enzyme to produce a mature library displaying cyclic structures of the peptide variants.   
     
     
         15 . The method of  claim 14 , wherein the PTM enzyme is selected from the group consisting of a carboxylate-amine ligase, a cyclase, a dehydrogenase, a decarboxylase, an epimerase, an hydroxylase, a peptidase, a dehydratase, a transferase, an esterase, an oxygenase and an isomerase. 
     
     
         16 . The method of  claim 14 , wherein the replicable genetic packages of the mature library are selected from the group consisting of phage particles, bacteria, and fungi. 
     
     
         17 . The method of  claim 16 , wherein the phage particles are bacteriophage selected from the group consisting of M13 phage, T4 phage, T7 phage, fd phage, and lambda phage. 
     
     
         18 . The method of  claim 16 , wherein the fungi are yeast.