Cloning, expression and characterization of recombinant sweet-protein thaumatin II using the methylotrophic yeast Pichia pastoris

Authors

  • Tetsuya Masuda,

    1. Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan; telephone: + 81-(0)774-38-3742; fax: + 81-(0)774-38-3743
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  • Shinobu Tamaki,

    1. Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan; telephone: + 81-(0)774-38-3742; fax: + 81-(0)774-38-3743
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  • Ryosuke Kaneko,

    1. Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan; telephone: + 81-(0)774-38-3742; fax: + 81-(0)774-38-3743
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  • Ritsuko Wada,

    1. Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan; telephone: + 81-(0)774-38-3742; fax: + 81-(0)774-38-3743
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  • Yuki Fujita,

    1. Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan; telephone: + 81-(0)774-38-3742; fax: + 81-(0)774-38-3743
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  • Alka Mehta,

    1. Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan; telephone: + 81-(0)774-38-3742; fax: + 81-(0)774-38-3743
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  • Naofumi Kitabatake

    1. Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan; telephone: + 81-(0)774-38-3742; fax: + 81-(0)774-38-3743
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Abstract

Thaumatin, an intensely sweet-tasting protein, was secreted by the methylotrophic yeast Pichia pastoris. The mature thaumatin II gene was directly cloned from Taq polymerase-amplified PCR products by using TA cloning methods and fused the pPIC9K expression vector that contains Saccharomyces cerevisiae prepro α-mating factor secretion signal. Several additional amino acid residues were introduced at both the N- and C-terminal ends by genetic modification to investigate the role of the terminal end region for elicitation of sweetness in the thaumatin molecule. The secondary and tertiary structures of purified recombinant thaumatin were almost identical to those of the plant thaumatin molecule. Recombinant thaumatin II elicited a sweet taste as native plant thaumatin II; its threshold value of sweetness to humans was around 50 nM, which is the same as that of plant thaumatin II. These results demonstrate that the functional expression of thaumatin II was attained by Pichia pastoris systems and that the N- and C-terminal regions of the thaumatin II molecule do not -play an important role in eliciting the sweet taste of thaumatin. © 2004 Wiley Periodicals, Inc.

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