A Strategy of Exon Shuffling for Making Large Peptide Repertoires Displayed on Filamentous Bacteriophage

• Published in: Proceedings of the National Academy of Sciences - PNAS 1996-07, Vol.93 (15), p.7761-7766
• Main Authors: Fisch, Igor, Kontermann, Roland E., Finnern, Ricarda, Hartley, Oliver, Soler-Gonzalez, Andres S., Griffiths, Andrew D., Winter, Greg
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antibodies; Bacteria; Bacteriophage P1; Bacteriophages; Base Sequence; DNA Primers; Enzyme-Linked Immunosorbent Assay; Enzymes; Escherichia coli; Escherichia coli - genetics; Evolution; Evolution, Molecular; Exons; Glucuronidase - antagonists & inhibitors; Glucuronidase - biosynthesis; Introns; Models, Genetic; Molecular Sequence Data; Mutagenesis, Site-Directed; Nucleic Acid Conformation; Peptides; Peptides - chemical synthesis; Peptides - chemistry; Peptides - pharmacology; Phosphatases; Plasmids; Polymerase Chain Reaction; Proteins; Recombinant Fusion Proteins - biosynthesis; Recombination, Genetic; RNA, Ribosomal - biosynthesis; RNA, Ribosomal - genetics; Tetrahymena thermophila; Tetrahymena thermophila - genetics
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.15.7761

It has been suggested that recombination and shuffling between exons has been a key feature in the evolution of proteins. We propose that this strategy could also be used for the artificial evolution of proteins in bacteria. As a first step, we illustrate the use of a self-splicing group I intron with inserted lox-Cre recombination site to assemble a very large combinatorial repertoire (> 1011 members) of peptides from two different exons. Each exon comprised a repertoire of 10 random amino acids residues; after splicing, the repertoires were joined together through a central five-residue spacer to give a combinatorial repertoire of 25-residue peptides. The repertoire was displayed on filamentous bacteriophage by fusion to the pIII phage coat protein and selected by binding to several proteins, including β -glucuronidase. One of the peptides selected against β -glucuronidase was chemically synthesized and shown to inhibit the enzymatic activity (inhibition constant: 17 nM); by further exon shuffling, an improved inhibitor was isolated (inhibition constant: 7 nM). Not only does this approach provide the means for making very large peptide repertoires, but we anticipate that by introducing constraints in the sequences of the peptides and of the linker, it may be possible to evolve small folded peptides and proteins.