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Applied Microbiology and Biotechnology

, Volume 77, Issue 3, pp 597–603 | Cite as

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

  • Norihiko Okochi
  • Michiko Kato-Murai
  • Tetsuya Kadonosono
  • Mitsuyoshi Ueda
Biotechnologically Relevant Enzymes and Proteins

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.

Keywords

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

Notes

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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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Norihiko Okochi
    • 1
  • Michiko Kato-Murai
    • 1
  • Tetsuya Kadonosono
    • 1
  • Mitsuyoshi Ueda
    • 1
  1. 1.Division of Applied Life Sciences, Graduate School of AgricultureKyoto UniversityKyotoJapan

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