A STRATEGY OF EXON SHUFFLING FOR MAKING LARGE PEPTIDE REPERTOIRES DISPLAYED ON FILAMENTOUS BACTERIOPHAGE

It has been suggested that recombination and shuffling between exons h as been a key feature in the evolution of proteins. We propose that th is strategy could also be used for the artificial evolution of protein s in bacteria. As a first step, we illustrate the use of a self-splici ng group I intron with inserted lox-Cre recombination site to assemble a very large combinatorial repertoire (>10(11) members) of peptides f rom two different exons. Each exon comprised a repertoire of 10 random amino acids residues; after splicing, the repertoires were joined tog ether through a central five-residue spacer to give a combinatorial re pertoire of 25-residue peptides. The repertoire was displayed on filam entous bacteriophage by fusion to the pm phage coat protein and select ed by binding to several proteins, including beta-glucuronidase. One o f the peptides selected against beta-glucuronidase was chemically synt hesized and shown to inhibit the enzymatic activity (inhibition consta nt: 17 nM); by further exon shuffling, an improved inhibitor was isola ted (inhibition constant: 7 nM). Not only does this approach provide t he means for making very large peptide repertoires, but we anticipate that by introducing constraints in the sequences of the peptides and o f the linker, it may be possible to evolve small folded peptides and p roteins.