A strategy of exon shuffling for making large peptide repertoires displayed on filamentous bacteriophage.
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 (> 10(11) 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 beta-glucuronidase. One of the peptides selected against beta-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.
Fisch
I
I
Medical Research Council Centre for Protein Engineering, Cambridge, United Kingdom.
Kontermann
R E
RE
Finnern
R
R
Hartley
O
O
Soler-Gonzalez
A S
AS
Griffiths
A D
AD
Winter
G
G
D000595:Amino Acid Sequence; D000818:Animals; D017101:Bacteriophage P1; D001483:Base Sequence; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D019143:Evolution, Molecular; D005091:Exons; D005966:Glucuronidase / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D007438:Introns; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D012335:RNA, Ribosomal / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic; D016808:Tetrahymena thermophila / Q000235:genetics
D017931:DNA Primers; D010455:Peptides; D012335:RNA, Ribosomal; C065096:RNA, ribosomal, 26S; D011993:Recombinant Fusion Proteins; D005966:Glucuronidase