Construction, exploitation and evolution of a new peptide library displayed at high density by fusion to the major coat protein of filamentous phage.
The amino-terminus of the major coat protein (PVIII) of filamentous phage can be extended, up to 6-7 residues, without interfering with the phage life cycle. We have constructed a library of approximately ten millions different phage each displaying a different octapeptide joined to the amino-terminus of the 2700 copies of PVIII. Most of the resulting clones are able to produce infective particles. This molecular repertoire constituted by the periodic regular decoration of the phage filament surface, can be utilized to search elements that bind proteins or relatively small organic molecules like the textile dye Cibacron blue. By sequential growth cycles we have performed a library evolution experiment to select phage clones that have a growth advantage in the absence of any requirement for binding a specific target. The consensus of the best growers reveals a Pro rich sequence with large hydrophobic residues at position 7 and Asn at position 1 of the random peptide insert. We propose that the assembly secretion process is favoured in phages displaying this family of peptides since they fit the groove between two adjacent PVIII subunits by making advantageous molecular contacts on the phage surface.
Iannolo
G
G
Department of Biology, University of Rome Tor Vergata, Italy.
Minenkova
O
O
Gonfloni
S
S
Castagnoli
L
L
Cesareni
G
G
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001483:Base Sequence; D001666:Binding Sites, Antibody; D002213:Capsid / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D017911:Inoviridae / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis; D014227:Triazines / Q000378:metabolism
D000911:Antibodies, Monoclonal; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014227:Triazines; C012842:Cibacron Blue F 3GA