Evolvability of random polypeptides through functional selection within a small library.
A directed evolution with phage-displayed random polypeptides of about 140 amino acid residues was followed until the sixth generation under a selection based on affinity to a transition state analog for an esterase reaction. The experimental design deliberately limits the observation to only 10 clones per generation. The first generation consists of three soluble random polypeptides and seven arbitrarily chosen clones from a previously constructed library. The clone showing the highest affinity in a generation was selected and subjected to random mutagenesis to generate variants for the next generation. Even within only 10 arbitrarily chosen polypeptides in each of the generations, there are enough variants in accord to capacity of binding affinity. In addition, the binding capacity of the selected polypeptides showed a gradual continuous increase over the generation. Furthermore, the purified selected random polypeptides exhibited a gradual but significant increase in esterase activity. The ease of the functional development within a small sequence variety implies that enzyme evolution is prompted even within a small population of random polypeptides.
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1, Yamada-oka, Suita Shi, Osaka, 565-0871, Japan.
Asao
A
Yamauchi
Toshihiro
T
Nakashima
Nobuhiko
N
Tokuriki
Masato
M
Hosokawa
Hideki
H
Nogami
Shingo
S
Arioka
Itaru
I
Urabe
Tetsuya
T
Yomo
D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000235:genetics; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015723:Gene Library; D014644:Genetic Variation; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009596:Nitrophenols / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity
D009596:Nitrophenols; D019151:Peptide Library; D010455:Peptides; C008642:4-nitrophenyl acetate; D004950:Esterases