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Design of a serine protease-like catalytic triad on an antibody light chain displayed on the yeast cell surface

Applied Microbiology and Biotechnology volume 77, pages 597–603 (2007)Cite this article

Abstract

Lc-WT, the wild-type light chain of antibody, and Lc-Triad, its double mutant with E1D and T27aS designing for the construction of catalytic triad within Asp1, Ser27a, and original His93 residues, were displayed on the cell surface of the protease-deficient yeast strain BJ2168. When each cell suspension was reacted with BODIPY FL casein and seven kinds of peptide-MCA substrates, respectively, a remarkable difference in hydrolytic activities toward Suc-GPLGP-MCA (succinyl-Gly-Pro-Leu-Gly-Pro-MCA), a substrate toward collagenase-like peptidase, was observed between the constructs: Lc-Triad-displaying cells showed higher catalytic activity than Lc-WT-displaying cells. The difference disappeared in the presence of the serine protease inhibitor diisopropylfluorophosphate, suggesting that the three amino acid residues, Ser27a, His93, and Asp1, functioned as a catalytic triad responsible for the proteolytic activity in a similar way to the anti-vasoactive intestinal peptide (VIP) antibody light chain. A serine protease-like catalytic triad (Ser, His, and Asp) is considered to be directly involved in the catalytic mechanism of the anti-VIP antibody light chain, which moderately catalyzes the hydrolysis of VIP. These results suggest the possibility of new approach for the creation of tailor-made proteases beyond limitations of the traditional immunization approach.

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Acknowledgment

The DNA encoding the 6D9 light chain gene was donated by Prof. I. Fujii, Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Japan.

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Affiliations

  1. Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan

    Norihiko Okochi, Michiko Kato-Murai, Tetsuya Kadonosono & Mitsuyoshi Ueda

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  1. Norihiko Okochi
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  2. Michiko Kato-Murai
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  3. Tetsuya Kadonosono
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  4. Mitsuyoshi Ueda
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Correspondence to Mitsuyoshi Ueda.

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Okochi, N., Kato-Murai, M., Kadonosono, T. et al. Design of a serine protease-like catalytic triad on an antibody light chain displayed on the yeast cell surface. Appl Microbiol Biotechnol 77, 597–603 (2007). https://doi.org/10.1007/s00253-007-1197-0

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  • DOI: https://doi.org/10.1007/s00253-007-1197-0

Keywords

  • Catalytic triad
  • Proteolytic antibody light chain
  • Cell surface display
  • Site-directed mutagenesis