A Novel Method of High Yield Cell-Free Protein Synthesis
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概要
- 論文の詳細を見る
A novel method of cell-free protein synthesis named PER (preincubation followed by energy replenishment was developed. An incubation mixture containing wheat germ extract (WGE), ATP, GTP, and amino acids was first preincubated at 30℃ for a certain time and then subjected to centrifugal ultrafiltration to remove most of the low molecular weight components. After filtration, an energy replenishment buffer and template mRNA were added to the filtrated incubation mixture and translation was carried out at 30℃ for 1 h. Protein synthesis started earlier and progressed more rapidly using the PER method in comparison with the conventional cell-free protein synthesis method : the rate of protein production and the amount of protein produced increased 8-fold and 12-fold at maximum, respectively. For luciferase production using WGE, the optimal preincubation time at 30℃ was found to be 30 min. The presence of energy biochemicals in the incubation mixture during preincubation significantly contributed to the increase in protein productivity, presumably because (i) the formation of certain complexes, such as the ternary complex (eIF-2・GTP・aminoacyl-tRNA) and the 43S preinitiation complex (ternary complex・40S subunit), requires energy biochemicals; and (ii) the formation of the 43S preinitiation complex accelerates the formation of the 48S preinitiation complex (43S preinitiation complex・mRNA) which is a rate-limiting step in the initiation of protein synthesis. Filtration of the incubation mixture by centrifugal ultrafiltration also played a major role in increasing the protein productivity, presumably because formation of the complexes was accelerated by the temporary condensation that occurred during filtration. Energy replenishment (ER), supplied by a replenishment buffer following ultrafiltration of the preincubated mixture, could restore the energy charge of the mixture to the mixture to the level required for protein synthesis. The PER method can be used as a highly efficient cell-free protein synthesis method for producing protein. ER is also a good method in itself for use in experimental studies on the mechanism of protein synthesis since it lessens the effects of energy decline.
- 社団法人日本生物工学会の論文
- 1997-12-25
著者
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Suzuki Eiji
Department of Internal Medicine II, Fukushima Medical University School of Medicine, Fukushima
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Terada Satoshi
Department of Applied Chemistry and Biotechnology, University of Fukui
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Suzuki E
Shinshu Univ. Ueda Jpn
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Terada Satoshi
Department Of Chemistry And Biotechnology Graduate School Of Engineering The University Of Tokyo
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Terada Satoshi
Department Of Applied Chemistry And Biotechnology Faculty Of Engineering University Of Fukui
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SUZUKI Eiji
TDK Corporation
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SHEN XIN-CHUN
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo
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Suzuki Eiji
Department Of Fine Materials Engineering Faculty Of Textile Science & Technology Shinshu Univers
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Shen Xin-chun
Department Of Chemistry And Biotechnology Graduate School Of Engineering The University Of Tokyo
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Terada Shoichi
Department Of Chemistry And Biotechnology Graduate School Of Engineering The University Of Tokyo
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YAO SHUILIANG
Research Institute of Innovative Technology for the Earth
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YAO SHUILIANG
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo
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Yao Shuiliang
Department Of Chemistry And Biotechnology Graduate School Of Engineering The University Of Tokyo
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Suzuki E
Tdk Corporation
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Suzuki Eiji
Department Of Breast Surgery Tokyo Metropolitan Komagome Hospital
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Suzuki Eiji
Department Of Biochemistry Faculy Of Veterinary Medicine Hokkaido University
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