無細胞タンパク質合成系ノ高度化ト応用

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Cell-free protein synthesis systems have been studied in order to expand their applications in the fields of biochemical and biomolecular engineering. (1) Biochemical analysis of the wheat germ translation system and Escherichia coli coupled transcription/translation system revealed that consumption of ATP and GTP caused an early halt to translation in cell-free systems. Modification of the ATP-regenerating system extensively prolonged the duration of translation, resulting in an increased amount of proteins. In addition, a novel reactor using a hollow-fiber membrane was constructed to increase the productivity of the E. coli cell-free system. (2) Extracellular proteins, such as a single-chain Fv, bacterial lipases, and phospholipase D, were synthesized in their active forms and aggregate formation was avoided by controlling the oxidation condition of the E. coli system. A hetero-oligomeric Fab fragment of antibody was also synthesized successfully. (3) In vitro combinatorial mutagenesis, which enables rapid construction of mutated proteins at multiple amino acid residues, was developed. Application of the system to a Burkholderia cepacia lipase resulted in the finding of a mutant enzyme showing greatly different substrate specificity. (4) A novel method was developed for constructing a protein library by combining PCR amplification of single DNA molecules encoding proteins (single-molecule PCR) and cell-free protein synthesis exclusively without using living cells. The new system, termed 'SIMPLEX', has been shown to produce a highly uniform protein library suitable for quantitative screening. The system provides a powerful tool for high-throughput generation and selection of proteins with novel functions.
公益社団法人日本生物工学会の論文
2003-02-25

無細胞タンパク質合成系ノ高度化ト応用

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