Design of Carrier tRNAs and Selection of Four-Base Codons for Efficient Incorporation of Various Nonnatural Amino Acids into Proteins in Spodoptera frugiperda 21 (Sf21) Insect Cell-Free Translation System(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNO

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概要

• 論文の詳細を見る

• Spodoptera frugiperda 21 (Sf21) insect cell-free protein synthesizing system was expanded to include nonnatural amino acids. Orthogonal tRNAs that work as carriers of nonnatural amino acids in the insect system were explored. Four-base codons for assigning the positions of nonnatural amino acids were also selected. Mutated streptavidin mRNAs that contained different four-base codons were prepared and added to the insect cell-free system in the presence of various tRNAs possessing the corresponding four-base anticodons. The tRNAs were chemically aminoacylated with various types of nonnatural amino acids to examine their incorporation efficiencies. Using p-nitrophenylalanine as the nonnatural amino acid and streptavidin as the target protein, tRNA sequences and the types of four-base codons were optimized to maximize the yield of the nonnatural mutant and to minimize production of full-length proteins that do not contain the nonnatural amino acid. Among the tRNA sequences taken from a variety of tRNAs of nonstandard structures, the tRNA derived from Methanosarcina acetivorans tRNA^<Pyl> was the most efficient and orthogonal tRNA. Of the CGGN-type four-base codons, CGGA and CGGG were the most efficient ones for assigning the positions of nonnatural amino acids. p-Nitrophenylalanine and 2-naphthylalanine were efficiently incorporated as in the case of Escherichia coli and rabbit reticulocyte cell-free systems. Much less efficient incorporation was observed, however, for other nonnatural amino acids, indicating that the insect system is less tolerant to the structural diversity of amino acids than the E. coli cell-free system.

• 社団法人日本生物工学会の論文
• 2006-12-25

著者

• OHTSUKI Takashi

  Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Yamanashi University

• Sisido Masahiko

  Department of Bioscience and Biotechnology, Okayama University

• Ohtsuki Takashi

  Department Of Applied Chemistry And Biotechnology Faculty Of Engineering University Of Yamanashi

• Ohtsuki Takashi

  Dep. Of Bioscience And Biotechnology Okayama Univ. 3-1-1 Tsushimanaka Okayama 700-8530 Jpn

• Taki Masumi

  Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University

• Tokuda Yasunori

  Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University

• Sisido Masahiko

  Dep. Of Bioscience And Biotechnology Okayama Univ. 3-1-1 Tsushimanaka Okayama 700-8530 Jpn

• Sisido Masahiko

  Department Of Bioscience And Biotechnology Okayama University

• Taki Masumi

  Department Of Bioscience And Biotechnology Faculty Of Engineering Okayama University

• Taki Masumi

  The Graduate School Of Natural Science And Technology Okayama University

• Sisido Masahiko

  Department Of Bioengineering Science Faculty Of Engineering Okayama University

• Tokuda Yasunori

  Department Of Bioscience And Biotechnology Faculty Of Engineering Okayama University

• Ohtsuki Takashi

  Department Of Applied Biology Faculty Of Textile Science And Technology Shinsyu University

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