Selection of peptides with antibody-like properties

Abstract

The present invention provides a highly sensitive screening assay for the identification of peptide binding partners to virtually any peptide or polypeptide ligand. Utilizing an expression-repression readout system, the inventors have screened libraries of peptides and identified relatively small peptide molecules that bind to the provided target.

1 . A method for identifying a peptide-peptide interaction comprising: 
 (a) providing a first fusion construct comprising target peptide fused to a first DNA binding domain;    (b) providing a second fusion construct comprising a library encoded peptide (LEP) fused to second DNA binding domain (DBD), wherein said second DBD works as a complex with said first DBD to facilitate binding of said complex to a prokaryotic operator region;    (c) contacting said first and second fusion constructs in a prokaryotic host cell which comprises said prokaryotic operator region, wherein said prokaryotic operator region is operationally linked to a coding region for one or more indicator polypeptides; and    (d) determining binding of said complex to said operator region,    whereby binding of said complex to said operator region identifies said LEP as a binding partner for said target peptide.    
     
     
         2 . The method of  claim 1 , wherein binding of said complex to said operator acts blocks the transcription of said coding region.  
     
     
         3 . The method of  claim 1 , wherein said one or more indicator polypeptides render said prokaryotic host cell insensitive to phage infection.  
     
     
         4 . The method of  claim 3 , wherein step (d) comprises infection with a phage that infects, replicates and lyses said prokaryotic host cell.  
     
     
         5 . The method of  claim 4 , wherein said operator is the lacZ operator, and the first and second DBDs are derived from the λ repressor.  
     
     
         6 . The method of  claim 1 , wherein one or more indicator polypeptides produce a calorimetric or fluorescent product.  
     
     
         7 . The method of  claim 1 , wherein said one or more indicator polypeptides is β-gal.  
     
     
         8 . The method of  claim 1 , wherein said target peptide is 5 to about 5000 residues in length.  
     
     
         9 . The method of  claim 1 , wherein said target peptide is 10 to about 2000 residues in length.  
     
     
         10 . The method of  claim 1 , wherein said LEP is 5 to about 50 residues.  
     
     
         11 . The method of  claim 1 , wherein said first and second fusion constructs are encoded by a nucleic acid segment under the control of a promoter operable in said prokaryotic host cell.  
     
     
         12 . The method of  claim 1 , wherein said target peptide and LEP bind with an affinity in the range of about 10 −3  to about 10 −6  M.  
     
     
         13 . The method of  claim 12 , wherein said target peptide and LEP bind with an affinity in the range of about 10 −4  M.  
     
     
         14 . The method of  claim 12 , wherein said target peptide and LEP bind with an affinity in the range of about 10 −5  M.  
     
     
         15 . The method of  claim 12 , wherein said target peptide and LEP bind with an affinity in the range of about 10 −6  M.  
     
     
         16 . The method of  claim 1 , further comprising random mutagenesis of said LEP, followed by measuring the change, if any, in the binding affinity of said LEP for said target.  
     
     
         17 . The method of  claim 16 , wherein said measuring comprises effecting binding of said LEP to said target peptide under conditions more stringent than in  claim 1 .  
     
     
         18 . The method of  claim 1 , further comprising: 
 (e) linking said identified LEP to a third peptide, whereby said linking permits said identified LEP and said third peptide to interact independently with said target peptide;    (f) then contacting said target peptide with the identified LEP-third peptide complex, and    (g) followed by determining the change, if any, in the binding affinity of said LEP for said target peptide.    
     
     
         19 . The method of  claim 18 , wherein said measuring comprises effecting binding of said LEP to said target peptide under conditions more stringent than in  claim 1 .  
     
     
         20 . The method of  claim 18 , wherein said third peptide is known to bind said target peptide.  
     
     
         21 . The method of  claim 18 , wherein said third peptide is a member of a peptide or peptidomimetic library.  
     
     
         22 . The method of  claim 1 , wherein said target peptide is an enzyme substrate, an antigen, or a eukaryotic cell antigen.  
     
     
         23 . The method of  claim 22 , wherein said target peptide is an enzyme substrate.  
     
     
         24 . The method of  claim 23 , wherein said enzyme substrate is bacterial, viral or fungal antigen.  
     
     
         25 . The method of  claim 22 , wherein said target peptide is a eukaryotic cell antigen.  
     
     
         26 . The method of  claim 25 , wherein said eukaryotic cell antigen is a tumor cell marker, an HLA antigen, a cell surface receptor, or a cell surface transporter.  
     
     
         27 . The method of  claim 1 , further comprising, prior to said determining, the step of stabilizing the interaction between said target peptide and said LEP.  
     
     
         28 . The method of  claim 27 , wherein said stabilizing is achieved via cross-linking or phototrapping.  
     
     
         29 . The method of  claim 1 , wherein said first peptide comprises a multimer of a smaller peptide unit.  
     
     
         30 . The method of  claim 1 , further comprising assessing binding of said target peptide to said identified LEP by Western blot, mass spectroscopy, or nuclear magnetic resonance.  
     
     
         31 . A method for screening a peptide library for peptide-peptide interactions comprising: 
 (a) providing a plurality of a first fusion construct comprising a target peptide fused to a first DNA binding domain;    (b) providing a plurality of second fusion construct comprising a library of encoded peptide (LEPs) fused to second DNA binding domain (DBD), wherein said second DBD works as a complex with said first DBD to facilitate binding of said complex to a prokaryotic operator region;    (c) transferring said pluralities of first and second fusion constructs into a prokaryotic host cell which comprises said prokaryotic operator region, wherein said prokaryotic operator region is operationally linked to a coding region for one or more indicator polypeptides; and    (d) determining binding of complexes to said operator region,    whereby binding of said complexes to said operator region identifies associated LEPs as binding partners for said target peptide.    
     
     
         32 . The method of  claim 3 , wherein steps (a)-(d) are repeated at least once using the LEP identified in step (d).  
     
     
         33 . The method of  claim 31 , wherein said LEPs are synthesized from a four base cutter-digested DNA library.  
     
     
         34 . The method of  claim 33 , further comprising the step of sequencing a DNA encoding an identified LEP.  
     
     
         35 . A library encoded peptide (LEP) selected according to a method comprising: 
 (a) providing a first fusion construct comprising target peptide fused to a first DNA binding domain;    (b) providing a second fusion construct comprising said LEP fused to second DNA binding domain (DBD), wherein said second DBD works as a complex with said first DBD to facilitate binding of said complex to a prokaryotic operator region;    (c) contacting said first and second fusion constructs in a prokaryotic host cell comprising said prokaryotic operator region, wherein said prokaryotic operator regions is operationally linked to a coding region for one or more indicator polypeptides; and    (d) determining binding of said complex to said operator region, 
 whereby binding of said complex to said operator region identifies said LEP as a binding partner for said target peptide.  
   
     
     
         36 . A heterodimeric binding molecule comprising: 
 (a) a first peptide that binds to a target molecule;    (b) a second peptide that binds to said target molecule,    wherein at least one of said first and second peptides is a member of a peptide library; and    (c) a linker molecule connecting said first and second peptides such that the linking permits said first and second peptides to interact independently with said target molecule.    
     
     
         37 . The binding molecule of  claim 36 , further comprising a moiety that permits recovery of said molecule.  
     
     
         38 . The binding molecule of  claim 37 , wherein said moiety is a magnetic bead.  
     
     
         39 . The binding molecule of  claim 36 , wherein said heterodimeric binding molecule is expressed on the surface of a phage.