Surface expression libraries of randomized peptides

Abstract

A composition of matter comprising a plurality of procaryotic cells containing a diverse population of expressible oligonucleotides operationally linked to expression elements, said expressible oligonucleotides having a desirable bias of random codon sequences.

I claim:  
     
         1 . A composition of matter comprising a plurality of cells containing a diverse population of expressible oligonucleotides operationally linked to expression elements, said expressible oligonucleotides having a desirable bias of random codon sequences produced from random combinations of first and second oligonucleotide precursor populations having a desirable bias of random codon sequences.  
     
     
         2 . The composition of    claim 1   , wherein the desirable bias of random codon sequences of said first and second oligonucleotides is unbiased.  
     
     
         3 . The composition of    claim 1   , wherein the desirable bias of random codon sequences of said first and second oligonucleotides is biased toward a predetermined sequence.  
     
     
         4 . The composition of    claim 1   , wherein said first and second oligonucleotides having random codon sequences have at least one specified codon at a predetermined position.  
     
     
         5 . The composition of    claim 1   , wherein said cells are procaryotes.  
     
     
         6 . The composition of    claim 1   , wherein said cells are  E. coli.    
     
     
         7 . A kit for the preparation of vectors useful for the expression of a diverse population of random peptides from combined first and second oligonucleotides having a desirable bias of random codon sequences, comprising: two vectors: a first vector having a cloning site for said first oligonucleotides and a pair of restriction sites for operationally combining first oligonucleotides with second oligonucleotides; and a second vector having a cloning site for said second oligonucleotides and a pair of restriction sites complementary to those on said first vector, one or both vectors containing expression elements capable of being operationally linked to said combined first and second oligonucleotides.  
     
     
         8 . The kit of    claim 7   , wherein said vectors are in a filamentous bacteriophage.  
     
     
         9 . The kit of    claim 8   , wherein said filamentous bacteriophage are M13.  
     
     
         10 . The kit of    claim 7   , wherein said vectors are plasmids.  
     
     
         11 . The kit of    claim 7   , wherein said vectors are phagemids.  
     
     
         12 . The kit of    claim 7   , wherein the desirable bias of random codon sequences of said first and second oligonucleotides is unbiased.  
     
     
         13 . The kit of    claim 7   , wherein the desirable bias of random codon sequences of said first and second oligonucleotides is diverse but biased toward a predetermined sequence.  
     
     
         14 . The kit of    claim 7   , wherein said first and second oligonucleotides having a desirable bias of random codon sequences have at least one specified codon at a predetermined position.  
     
     
         15 . The kit of    claim 7   , wherein said pair of restriction sites are Fok I.  
     
     
         16 . A cloning system for expressing random peptides from diverse populations of combined first and second oligonucleotides having a desirable bias of random codon sequences, comprising: a set of first vectors having a diverse population of first oligonucleotides having a desirable bias of random codon sequences and a set of second vectors having a diverse population of second oligonucleotides having a desirable bias of random codon sequences, said first and second vectors each having a pair of restriction sites so as to allow the operational combination of first and second oligonucleotides into a contiguous oligonucleotide having a desirable bias of random codon sequences.  
     
     
         17 . The cloning system of    claim 16   , wherein the desirable bias of random codon sequences of said first and second oligonucleotides is unbiased.  
     
     
         18 . The cloning system of    claim 16   , wherein the desirable bias of random codon sequences of said first and second oligonucleotides is diverse but biased toward a predetermined sequence.  
     
     
         19 . The cloning system of    claim 16   , wherein said first and second oligonucleotides having a desirable bias of random codon sequences have at least one specified codon at a predetermined position.  
     
     
         20 . The cloning system of    claim 16   , wherein said combined first and second vectors is through a pair of restriction sites.  
     
     
         21 . The cloning system of    claim 16   , wherein said pair of restriction sites are Fok I.  
     
     
         22 . A composition of matter comprising a plurality of cells containing a diverse population of expressible oligonucleotides operationally linked to expression elements, said expressible oligonucleotides having a desirable bias of random codon sequences.  
     
     
         23 . The composition of    claim 22   , wherein said cells are procaryotes.  
     
     
         24 . The composition of    claim 22   , wherein said expressible oligonucleotides are expressed as peptide fusion proteins on the surface of a filamentous bacteriophage.  
     
     
         25 . The composition of    claim 22   , wherein said filamentous bacteriophage is M13.  
     
     
         26 . The composition of    claim 22   , wherein said fusion protein contains the product of gene VIII.  
     
     
         27 . The composition of    claim 22   , wherein said diverse population of oligonucleotides having a desirable bias of random codon sequences are produced from the combination of diverse populations of first and second oligonucleotides having a desirable bias of random codon sequences.  
     
     
         28 . The composition of    claim 22   , wherein the desirable bias of random codon sequences of said oligonucleotides is unbiased.  
     
     
         29 . The composition of    claim 22   , wherein the desirable bias of random codon sequences of said oligonucleotides is diverse but biased toward a predetermined sequence.  
     
     
         30 . The composition of    claim 22   , wherein said oligonucleotides having a desirable bias of random codon sequences have at least one specified codon at a predetermined position.  
     
     
         31 . A plurality of vectors containing a diverse population of expressible oligonucleotides having a desirable bias of random codon sequences.  
     
     
         32 . The vectors of    claim 31   , wherein said oligonucleotides are expressible as fusion proteins on the surface of filamentous bacteriophage.  
     
     
         33 . The vectors of    claim 31   , wherein said filamentous bacteriophage is M13.  
     
     
         34 . The vectors of    claim 31   , wherein said fusion protein contains the product of gene VIII.  
     
     
         35 . The vectors of    claim 31   , wherein the desirable bias of random codon sequences of said oligonucleotides is unbiased.  
     
     
         36 . The vectors of    claim 31   , wherein the desirable bias of random codon sequences of said oligonucleotides is diverse but biased toward a predetermined sequence.  
     
     
         37 . The vectors of    claim 31   , wherein said oligonucleotides having a desirable bias of random codon sequences have at least one specified codon at a predetermined position.  
     
     
         38 . A composition of matter, comprising a diverse population of oligonucleotides having a desirable bias of random codon sequences produced from random combinations of two or more oligonucleotide precursor populations having a desirable bias of random codon sequences.  
     
     
         39 . A method of constructing a diverse population of vectors having combined first and second oligonucleotides having a desirable bias of random codon sequences capable of expressing said combined oligonucleotides as random peptides, comprising the steps of: 
 (a) operationally linking sequences from a diverse population of first oligonucleotides having a desirable bias of random codon sequences to a first vector;    (b) operationally linking sequences from a diverse population of second oligonucleotides having a desirable bias of random codon sequences to a second vector; and    (c) combining the vector products of steps (a) and (b) under conditions where said populations of first and second oligonucleotides are joined together into a population of combined vectors capable of being expressed.    
     
     
         40 . The method of    claim 39   , wherein the desirable bias of random codon sequences of said first and second oligonucleotides is unbiased.  
     
     
         41 . The method of    claim 39   , wherein the desirable bias of random codon sequences of said first and second oligonucleotides is diverse but biased toward a predetermined sequence.  
     
     
         42 . The method of    claim 39   , wherein said first and second oligonucleotides having a desirable bias of random codon sequences have at least one specified codon at a predetermined position.  
     
     
         43 . The method of    claim 38   , wherein steps (a) through (c) are repeated two or more times.  
     
     
         44 . A method of selecting a peptide capable of being bound by a ligand binding protein from a population of random peptides, comprising: 
 (a) operationally linking a diverse population of first oligonucleotides having a desirable bias of random codon sequences to a first vector;    (b) operationally linking a diverse population of second oligonucleotides having a desirable bias of random codon sequences to a second vector;    (c) combining the vector products of steps (a) and (b) under conditions where said populations of first and second oligonucleotides are joined together into a population of combined vectors;    (d) introducing said population of combined vectors into a compatible host under conditions sufficient for expressing said population of random peptides; and    (e) determining the peptide which binds to said ligand binding protein.    
     
     
         45 . The method of    claim 44   , wherein the desirable bias of random codon sequences of said first and second oligonucleotides is unbiased.  
     
     
         46 . The method of    claim 44   , wherein the desirable bias of random codon sequences of said first and second oligonucleotides is diverse but biased toward a predetermined sequence.  
     
     
         47 . The method of    claim 44   , wherein said first and second oligonucleotides having a desirable bias of random codon sequences have at least one specified codon at a predetermined position.  
     
     
         48 . The method of    claim 44   , wherein steps (a) through (c) are repeated two or more times.  
     
     
         49 . A method for determining the nucleic acid sequence encoding a peptide capable of being bound by a ligand binding protein which is selected from a population of random peptides, comprising: 
 (a) operationally linking a diverse population of first oligonucleotides having a desirable bias of random codon sequences to a first vector;    (b) operationally linking a diverse population of second oligonucleotides having a desirable bias of random codon sequences to a second vector;    (c) combining the vector products of steps (a) and (b) under conditions where said populations of first and second oligonucleotides are joined together into a population of combined vectors;    (d) introducing said population of combined vectors into a compatible host under conditions sufficient for expressing said population of random peptides;    (e) determining the peptide which binds to said ligand binding protein;    (f) isolating the nucleic acid encoding said peptide; and    (g) sequencing said nucleic acid.    
     
     
         50 . The method of    claim 49   , wherein the desirable bias of random codon sequences of said first and second oligonucleotides is unbiased.  
     
     
         51 . The method of    claim 49   , wherein the desirable bias of random codon sequences of said first and second oligonucleotides is diverse but biased toward a predetermined sequence.  
     
     
         52 . The method of    claim 49   , wherein said first and second oligonucleotides having a desirable bias of random codon sequences have at least one specified codon at a predetermined position.  
     
     
         53 . The method of    claim 49   , wherein steps (a) through (c) are repeated two or more times.  
     
     
         54 . A method of constructing a diverse population of vectors containing expressible oligonucleotides having a desirable bias of random codon sequences, comprising operationally linking a diverse population of oligonucleotides having a desirable bias of random codon sequences to expression elements.  
     
     
         55 . The method of    claim 54   , wherein said oligonucleotides are expressible as fusion proteins on the surface of filamentous bacteriophage.  
     
     
         56 . The method of    claim 54   , wherein said filamentous bacteriophage are M13.  
     
     
         57 . The method of    claim 54   , wherein said fusion protein contains the product of gene VIII.  
     
     
         58 . The method of    claim 54   , wherein the desirable bias of random codon sequences of said oligonucleotides is unbiased.  
     
     
         59 . The method of    claim 54   , wherein the desirable bias of random codon sequences of said oligonucleotides is diverse but biased toward a predetermined sequence.  
     
     
         60 . The method of    claim 54   , wherein said oligonucleotides having a desirable bias of random codon sequences have at least one specified codon at a predetermined position.  
     
     
         61 . The method of    claim 54   , wherein said operationally linking further comprising the steps of: 
 (a) operationally linking a diverse population of first oligonucleotides having a desirable bias of random codon sequences to a first vector;    (b) operationally linking a diverse population of second oligonucleotides having a desirable bias of random codon sequences to a second vector; and    (c) combining the vector products of steps (a) and (b) under conditions where said populations of first and second oligonucleotides are joined together into a population of combined vectors.    
     
     
         62 . The method of    claim 61   , wherein steps (a) through (c) are repeated two or more times.  
     
     
         63 . A method of selecting a peptide capable of being bound by a binding protein from a population of random peptides, comprising: 
 (a) operationally linking a diverse population of oligonucleotides having a desirable bias of random codon sequences to expression elements;    (b) introducing said population of vectors into a compatible host under conditions sufficient for expressing said population of random peptides; and    (c) determining the peptide which binds to said ligand binding protein.    
     
     
         64 . The method of    claim 63   , wherein said population of random peptides are expressed as fusion proteins on the surface of filamentous bacteriophage.  
     
     
         65 . The method of    claim 63   , wherein said filamentous bacteriophage are M13.  
     
     
         66 . The method of    claim 63   , wherein said fusion protein contains the product of gene VIII.  
     
     
         67 . The method of    claim 63   , wherein the desirable bias of random codon sequences of said oligonucleotides is unbiased.  
     
     
         68 . The method of    claim 63   , wherein the desirable bias of random codon sequences of said oligonucleotides is diverse but biased toward a predetermined sequence.  
     
     
         69 . The method of    claim 63   , wherein said oligonucleotides having a desirable bias of random codon sequences have at least one specified codon at a predetermined position.  
     
     
         70 . The method of    claim 63   , wherein step (a) further comprises: 
 (a1) operationally linking a diverse population of first oligonucleotides having a desirable bias of random codon sequences to a first vector;    (a2) operationally linking a diverse population of second oligonucleotides having a desirable bias of random codon sequences to a second vector; and    (a3) combining the vector products of steps (a) and (b) under conditions where said populations of first and second oligonucleotides are joined together into a population of combined vectors.    
     
     
         71 . The method of    claim 70   , wherein steps (a1) through (a3) are repeated two or more times.  
     
     
         72 . A method of determining the nucleic acid sequence encoding a peptide capable of being bound by a ligand binding protein which is selected from a population of random peptides, comprising: 
 (a) operationally linking a diverse population of oligonucleotides having a desirable bias of random codon sequences to expression elements.    (b) introducing said population of vectors into a compatible host under conditions sufficient for expressing said population of random peptides;    (c) determining the peptide which binds to said ligand binding protein;    (d) isolating the nucleic acid encoding said peptide; and    (e) sequencing said nucleic acid.    
     
     
         73 . The method of    claim 72   , wherein said population of random peptides are expressed as fusion proteins on the surface of filamentous bacteriophage.  
     
     
         74 . The method of    claim 72   , wherein said filamentous bacteriophage are M13.  
     
     
         75 . The method of    claim 72   , wherein said fusion protein contains the product of gene VIII.  
     
     
         76 . The method of    claim 72   , wherein the desirable bias of random codon sequences of said oligonucleotides is unbiased.  
     
     
         77 . The method of    claim 72   , wherein the desirable bias of random codon sequences of said oligonucleotides is diverse but biased toward a predetermined sequence.  
     
     
         78 . The method of    claim 72   , wherein said oligonucleotides having a desirable bias of random codon sequences have at least one specified codon at a predetermined position.  
     
     
         79 . The method of    claim 72   , wherein step (a) further comprises: 
 (a1) operationally linking a diverse population of first oligonucleotides having a desirable bias of random codon sequences to a first vector;    (a2) operationally linking a diverse population of second oligonucleotides having a desirable bias of random codon sequences to a second vector; and    (a3) combining the vector products of steps (a) and (b) under conditions where said populations of first and second oligonucleotides are joined together into a population of combined vectors.    
     
     
         80 . The method of    claim 78   , wherein steps (a1) through (a3) are repeated two or more times.  
     
     
         81 . A vector comprising two copies of a gene encoding a filamentous bacteriophage coat protein, both copies encoding substantially the same amino acid sequence but having different nucleotide sequences.  
     
     
         82 . The vector of    claim 81   , wherein said filamentous bacteriophage is M13.  
     
     
         83 . The vector of    claim 81   , wherein said gene is gene VIII.  
     
     
         84 . The vector of    claim 81   , wherein said vector has substantially the sequence shown in FIG. 5 (SEQ ID NO: 1).  
     
     
         85 . A vector comprising two copies of a gene encoding a filamentous bacteriophage coat protein, one copy of said gene capable of being operationally linked to an oligonucleotide wherein said oligonucleotide can be expressed as a fusion protein on the surface of said filamentous bacteriophage or as a soluble peptide.  
     
     
         86 . The vector of    claim 84   , wherein said one copy of said gene is expressed on the surface of said filamentous bacteriophage.  
     
     
         87 . The vector of    claim 84   , wherein said bacteriophage coat protein is M13 gene VIII.