Selection of a biotin protein ligase by phage display using a combination of in vitro selection and in vivo enzymatic activity.
We report the development of a phage display-based method for the direct selection of enzymatic activity by a combination of selection in vitro and an assay of enzymatic activity in vivo. We describe the selection of a biotin protein ligase (BPL) that specifically recognizes neurotensin as a substrate, as an example of the utility of our method. We constructed an enzyme library with a diversity of 8.8 x 10(6) by insertion of four randomized regions into a BPL backbone and used it for the selection of variant BPLs. We obtained an active BPL, which was identical to wild-type BPL, using the combination of selection in vitro and an assay of enzymatic activity in vivo. Our results indicate that our method is suitable for the selection of enzymes with specific functions of interest.
Iwamoto
Masahiro
M
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan.
Taki
Takashi
T
Fujita
Satoshi
S
D000595:Amino Acid Sequence; D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation; D003001:Cloning, Molecular; D008025:Ligases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009496:Neurotensin / Q000378:metabolism; D019151:Peptide Library
D019151:Peptide Library; D009496:Neurotensin; D001710:Biotin; D008025:Ligases