Selection of a biotin protein ligase by phage display using a combination of in vitro selection and in vivo enzymatic activity(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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概要

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• 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 8.8×10^6 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.

• 公益社団法人日本生物工学会の論文

著者

• Fujita Satoshi

  Department Of Bioengineering School Of Engineering The University Of Tokyo:research Institute For Ce

• Iwamoto Masahiro

  Department Of Chemistry And Biotechnology Graduate School Of Engineering The University Of Tokyo

• Taki Takashi

  Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo

• Taki Takashi

  Department Of Chemistry And Biotechnology Graduate School Of Engineering The University Of Tokyo

• Fujita Satoshi

  Department Of Chemistry And Biotechnology Graduate School Of Engineering The University Of Tokyo:res

• Fujita Satoshi

  Department Of Anesthesiology School Of Medicine Sapporo Medical University

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