Stabilized bioactive peptides and methods of identification, synthesis, and use

Abstract

An intracellular selection system allows screening for peptide bioactivity and stability. Randomized recombinant peptides are screened for bioactivity in a tightly regulated expression system, preferably derived from the wild-type lac operon. Bioactive peptides thus identified are inherently protease- and peptidase-resistant. Also provided are bioactive peptides stabilized by a stabilizing group at the N-terminus, the C-terminus, or both. The stabilizing group can be a small stable protein, such as the Rop protein, glutathione sulfotransferase, thioredoxin, maltose binding protein, or glutathione reductase, an α-helical moiety, or one or more proline residues.

What is claimed is: 
     
         1 . A plurality of vectors, wherein each said vector comprises a nucleic acid sequence encoding a polypeptide, said polypeptide comprising a randomized peptide and a first stabilizing group coupled to one of said randomized peptide's termini, wherein said first stabilizing group lacks the capacity to participate in the formation of an intramolecular disulfide bond within the polypeptide, and wherein said plurality of vectors comprises at least two different vectors, each of said at least two vectors encoding a different polypeptide. 
     
     
         2 . The plurality of vectors of  claim 1 , wherein said polypeptide further comprises a second stabilizing group coupled to the other terminus of said randomized peptide. 
     
     
         3 . The plurality of vectors of  claim 1 , wherein each said vector further comprises a tightly regulable expression control sequence operably linked to said nucleic acid sequence encoding said polypeptide. 
     
     
         4 . The plurality of vectors of  claim 1 , wherein said plurality comprises at least 50 different vectors, each of said at least 50 vectors encoding a different polypeptide. 
     
     
         5 . The plurality of vectors of  claim 1 , wherein said plurality comprises at least 1×10 6  different vectors, each of said at least 1×10 6  vectors encoding a different polypeptide. 
     
     
         6 . A plurality of host cells collectively containing the plurality of vectors of  claim 1 . 
     
     
         7 . The host cells of  claim 6 , wherein said host cells are prokaryotic. 
     
     
         8 . The host cells of  claim 6 , wherein said host cells are eukaryotic. 
     
     
         9 . A method for selecting bioactive peptides, said method comprising:
 a) expressing a plurality of stabilized polypeptides, wherein each said stabilized polypeptide is encoded by a different vector, and wherein each said stabilized polypeptide comprises a randomized peptide and a first stabilizing group coupled to one of said randomized peptide's termini, wherein said first stabilizing group lacks the capacity to participate in the formation of an intramolecular disulfide bond within the polypeptide, and wherein said plurality of stabilized polypeptide comprises at least two different polypeptides; and   b) screening each said stabilized polypeptide of said plurality for a bioactivity; and   c) selecting said bioactive peptides with said bioactivity.   
     
     
         10 . The method of  claim 9 , wherein said stabilized polypeptide further comprises a second stabilizing group coupled to the other terminus of said randomized peptide. 
     
     
         11 . The method of  claim 9 , wherein said plurality of stabilized polypeptides is expressed in eukaryotic cells. 
     
     
         12 . The method of  claim 11 , wherein said eukaryotic cells are cancer cells and said bioactivity is a complete or partial inhibition of cell division, induction of apoptosis, or cell toxicity. 
     
     
         13 . The method of  claim 12 , said method further comprising testing said selected bioactive peptides for bioactivity in normal cells compared with said cancer cells. 
     
     
         14 . The method of  claim 11 , wherein said eukaryotic cells are stem cells or cord blood cells and said bioactivity is an increase in cell growth rate.