Design of a serine protease-like catalytic triad on an antibody light chain displayed on the yeast cell surface |
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Authors: | Norihiko Okochi Michiko Kato-Murai Tetsuya Kadonosono Mitsuyoshi Ueda |
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Institution: | (1) Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan |
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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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Keywords: | Catalytic triad Proteolytic antibody light chain Cell surface display Site-directed mutagenesis |
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