HYBRID ROP-PIII PROTEINS FOR THE DISPLAY OF CONSTRAINED PEPTIDES ON FILAMENTOUS PHAGE CAPSIDS

In order to increase the versatility of phage display technology, it i s desirable to be able to impose some structural constraints on the pe ptides that are presented by the phage particles. This is currently no t feasible since the conformation of the capsid proteins, used to link the foreign peptide to the phage, are either unknown (pIII) or too si mple (pVIII) to permit the engineering of peptide inserts into a const rained context. To reach this scope we have modified the amino-terminu s of gene III by appending a well-characterized protein motif, the fou r-helix bundle of the bacterial protein Rop. Phage particles displayin g Rop can be separated from wild-type (wt) particles by affinity purif ication with an antibody. Rop can be extensively modified by substitut ing its solvent-exposed residues and/or by inserting peptides either i nto the carboxy-terminal tail or into the bend region that connects th e two alpha-helices of the monomer. These results open the possibility to construct peptide libraries where the peptides are constrained eit her into an OMEGA-loop type conformation or an alpha-helix. Libraries formed by peptides inserted into the carboxy-terminus can also be cons tructed. Furthermore, the system that we have developed permits to pro duce large quantities of the elements of the libraries in the cytoplas m or to display them on the capsid of filamentous phages.