Effective selection system for experimental evolution of random polypeptides towards DNA-binding protein.
An experimental evolution with selection based on binding affinity to DNA was carried out on a library of phage-displayed random polypeptides of about 140 amino acid residues. First, we constructed a system to artificially evolve phage-displayed random polypeptides toward binding to a target DNA containing a restriction enzyme site, in which random polypeptides capable of binding the DNA were recovered as complexes with the target DNA by digestion with the restriction enzyme. The experimental evolution cycle, including the above selection system and random mutagenesis for generating the next mutant library, was repeated until the fourth generation. The ability to bind to the DNA was enhanced per generation. In the fourth generation, convergence of the selected clones to a dominant sequence was observed. These results indicate that the newly constructed selection system is effective for exploring the evolvability of random polypeptides towards DNA-binding proteins.
Nakashima
Toshihiro
T
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Toyota
Hitoshi
H
Urabe
Itaru
I
Yomo
Tetsuya
T
D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D008969:Molecular Sequence Data; D019151:Peptide Library
D004268:DNA-Binding Proteins; D019151:Peptide Library