Molecular libraries

Abstract

The present invention provides a method of screening a library of peptides of formula M-G/M/V—(X) n wherein n is an integer from 3 to 18, M is methionine, G is glycine, V is valine and each X, which may be the same or different, is any genetically encoded amino acid, which method comprises: a) transforming a host cell population with a library of nucleic acid constructs which can express free peptides of formula M-G/M/V—(X) n ; b) culturing the transformed host cells under conditions suitable for intra-cellular expression of the peptides of formula M-G/M/V—(X) n ; and c) analysing the host cell population to determine the effect of the peptides of formula M-G/M/V—(X) n on a reporter system. Also describe are a library of nucleic acid constructs which can express free peptides in an intra-cellular environment, said peptides having the sequence M-G/M/V—(X) n , wherein n is an integer from 3 to 18, M is methionine, G is glycine, V is valine and each X, which may be the same or different, is any genetically encoded amino acid; and a method of generating a library of nucleic acid constructs which can express free peptides in an intra-cellular environment, which method comprises synthesising a library of DNA molecules which include the nucleotide sequence ATGGGA(NNK) n , wherein n is an integer from 3 to 18, N is A, C, T or G and K is G or T and wherein each NNK triplet may be the same or different, and inserting a library of synthesised DNA fragments which each includes a nucleotide sequence of formula ATGGGA(NNK) n into expression vectors.

1 . A method of screening a library of peptides of formula M-G/M/V—(X) n  wherein n is an integer from 3 to 18, M is methionine, G is glycine, V is valine and each X, which may be the same or different, is any genetically encoded amino acid, which method comprises: 
 a) transforming a host cell population with a library of nucleic acid constructs that expresses free peptides of formula M-G/M/V—(X) n ;    b) culturing the transformed host cells under conditions suitable for intracellular expression of the peptides of formula M-G/M/V—(X) n ; and    c) analyzing the host cell population to determine the effect of the peptides of formula M-G/M/V—(X) n  on a reporter system.    
     
     
         2 - 27 . (canceled)  
     
     
         28 . The method of  claim 1  wherein n is an integer from 3 to 5.  
     
     
         29 . The method of  claim 1  wherein the reporter system includes a target protein.  
     
     
         30 . The method of  claim 29  wherein the target protein is a nucleic acid binding protein.  
     
     
         31 . The method of  claim 30  wherein the nucleic acid binding protein is p53.  
     
     
         32 . The method of  claim 1  wherein the reporter system comprises a reporter gene.  
     
     
         33 . The method of  claim 32  wherein the reporter gene is operably linked to a sequence of nucleotides that provides a binding site for a target protein or for a protein that associates with or is a substrate for a target protein.  
     
     
         34 . The method of  claim 33  wherein the reporter gene is operably linked to a p21 or Bax promoter.  
     
     
         35 . The method of  claim 32  wherein the protein product of the reporter gene includes a secretion signal peptide.  
     
     
         36 . The method of  claim 32  wherein the protein product of the reporter gene includes a transmembrane domain.  
     
     
         37 . The method of  claim 32  wherein the host cells have been transfected with the reporter gene.  
     
     
         38 . The method of  claim 1  wherein the peptide library has at least 500 different members.  
     
     
         39 . The method of  claim 1  wherein the host cells are eukaryotic cells.  
     
     
         40 . A method of identifying a peptide ligand of formula M-G/M/V—(X) n  wherein n is an integer from 3 to 18, M is methionine, G is glycine, V is valine, and each X, which may be the same or different, is any genetically encoded amino acid, having a desired activity on a target protein, which method comprises: 
 a) transforming a host cell population with a library of nucleic acid constructs which expresses peptides of formula M-G/M/V—(X) n ;    b) culturing the transformed host cells under conditions suitable for intra-cellular expression of the peptides of formula M-G/M/V—(X) n ;    c) analyzing the host cell population to determine the effect of the expressed peptides on the target protein, wherein the target protein forms part of a reporter system; and    d) identifying the peptide of formula M-G/M/V—(X) n  in those cells in which the reporter system indicates a positive response.    
     
     
         41 . The method of  claim 40  wherein n is an integer from 3 to 5.  
     
     
         42 . The method of  claim 40  wherein the reporter system comprises a reporter gene.  
     
     
         43 . The method of  claim 42  wherein the reporter gene is operably linked to a sequence of nucleotides which provides a binding site for a target protein or for a protein which associates with or is a substrate for a target protein.  
     
     
         44 . The method of  claim 43  wherein the reporter gene is operably linked to a p21 or Bax promoter.  
     
     
         45 . The method of  claim 42  wherein the protein product of the reporter gene includes a secretion signal peptide.  
     
     
         46 . The method of  claim 42  wherein the protein product of the reporter gene includes a transmembrane domain.  
     
     
         47 . The method of  claim 42  wherein the host cells have been transfected with reporter gene.  
     
     
         48 . The method of  claim 40  wherein the peptide library has at least 500 different members.  
     
     
         49 . The method of  claim 40  wherein the host cells are eukaryotic cells.  
     
     
         50 . A library of nucleic acid constructs which expresses free peptides in an intra-cellular environment, the peptides having the sequence M-G/M/V—(X) n , wherein n is an integer from 3 to 18, M is methionine, G is glycine, V is valine and each X, which may be the same or different, is any genetically encoded amino acid.  
     
     
         51 . The library of  claim 50  wherein each nucleic acid construct contains a promoter region which is operably linked to the sequence which encodes the peptide of formula M-G/M/V—(X) n .  
     
     
         52 . The library of  claim 51  wherein the nucleic acid constructs are in the form of expression vectors.  
     
     
         53 . The library of  claim 52  wherein the expression vectors are capable of autonomous replication.  
     
     
         54 . The library of  claim 50  wherein n is an integer from 3 to 10.  
     
     
         55 . The library of  claim 50  which has at least 500 different members.  
     
     
         56 . The library of  claim 55  which has at least 2000 different members.  
     
     
         57 . The library of  claim 50  wherein the value of n is the same for each member of the library.  
     
     
         58 . A library of peptides, each member of the library having the amino acid sequence M-G/M/V—(X) n , wherein n is an integer from 3 to 18, M is methionine, G is glycine, V is valine and each X, which may be the same or different, is any genetically encoded amino acid.  
     
     
         59 . The library of  claim 58  wherein n is an integer from 3 to 10.  
     
     
         60 . The library of  claim 59  wherein n is an integer from 3 to 5.  
     
     
         61 . The library of  claim 58  which has at least 500 different members.  
     
     
         62 . The library of  claim 61  which has at least 2000 different members.  
     
     
         63 . The library of  claim 58  wherein the value of n is the same for each member of the library.  
     
     
         64 . A method of generating a library of nucleic acid constructs which expresses free peptides in an intracellular environment, which method comprises synthesizing a library of DNA molecules which include the nucleotide sequence ATGGGA (NNK) n , wherein n is an integer from 3 to 18, N is A, C, T or G and K is G or T and wherein each NNK triplet may be the same or different, and inserting a library of synthesized DNA fragments which each includes a nucleotide sequence of formula ATGGGA (NNK) n  into expression vectors.  
     
     
         65 . The method of  claim 64  wherein during synthesis of the library of DNA molecules equimolar amounts of nucleotides A, C, T and G are added for incorporation at each N position and equimolar amounts of nucleotides G and T are added for incorporation at each K position.  
     
     
         66 . The library of nucleic acid constructs prepared according to a method of  claim 64 .  
     
     
         67 . The library of nucleic acid constructs prepared according to a method of  claim 65.