Biotechnology and Bioprocess Engineering:BBE

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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In Vitro Combinatorial Mutagenesis of the 65th and 222nd Positions of the Green Fluorescent Protein of Aequarea victoria

  • Nakano, Hideo (Laboratory of Molecular Biotechnology, Graduate School of Biological and Agricultural Sciences, Nagoya University) ;
  • Okumura, Reiko (Laboratory of Molecular Biotechnology, Graduate School of Biological and Agricultural Sciences, Nagoya University) ;
  • Goto, Chinatsu (Laboratory of Molecular Biotechnology, Graduate School of Biological and Agricultural Sciences, Nagoya University) ;
  • Yamane, Tsuneo (Laboratory of Molecular Biotechnology, Graduate School of Biological and Agricultural Sciences, Nagoya University)
  • Published : 2002.09.01
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Abstract

By the in vitro combinatorial mutagenesis, which is a sequential reaction of PCR mutagenesis and in vitro coupled transcription/translation with Escherichia coli S30 extract, S65 and E222 of green fluorescent protein of Aequarea victoria were comprehensively changed to all possible combinations of amino acids, thus totally 400 mutant (including a wild type) proteins were simultaneously produced and their fluorescent properties were analyzed. Although a few mutations had been reported so far at the 222nd position, replacement E222 to all other19 amino acids gave fluorescent signal to the mutants by changing Ser 65 to Ala together. Among the mutants, replacement to G, A, S, Q, H and C gave relatively high fluorescence. The in vitro combinatorial mutagenesis, therefore, has been proved valuable for comprehensive structure-function studies of proteins.

Keywords

References

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