PROTEOLYTIC SELECTION FOR PROTEIN-FOLDING USING FILAMENTOUS BACTERIOPHAGES

Background: Filamentous bacteriophages have been used for the selectio n of folded peptide and protein 'ligands' by binding the phage to 'rec eptor'-coated solid phase. Here, using proteolysis, we have developed a technique for the selection of folded and stable proteins that is in dependent of their binding activities. Results: When a 21-residue pept ide comprising a protease cleavage site was introduced into the flexib le linker between the second and third domains of the minor coat prote in p3 of filamentous bacteriophage, the phages could be cleaved by try psin and were rendered non-infective. By contrast, phages displaying m utant barnases at this site were resistant to proteolysis, but were cl eaved and their infectivity was destroyed as the temperature was raise d. By mixing phages bearing two barnase mutants of differing stability , and adding protease at a temperature at which one mutant was resista nt and the other was sensitive, we were able to enrich by 1.6 x 10(4)- fold for phages bearing the more stable barnase. Conclusions: The appr oach provides a means for the selection of folded and stable proteins, and may be applicable to the selection of de novo proteins.