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Efficient coupled transcription/translation from PCR template by a hollow‐fiber membrane bioreactor
Author(s) -
Nakano Hideo,
Shinbata Tomoya,
Okumura Reiko,
Sekiguchi Satoshi,
Fujishiro Masatoshi,
Yamane Tsuneo
Publication year - 1999
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19990720)64:2<194::aid-bit8>3.0.co;2-5
Subject(s) - bioreactor , t7 rna polymerase , rna polymerase , transcription (linguistics) , protein biosynthesis , chemistry , escherichia coli , polymerase chain reaction , polymerase , terminator (solar) , gene , microbiology and biotechnology , biology , biochemistry , bacteriophage , organic chemistry , linguistics , philosophy , ionosphere , physics , astronomy
A novel bioreactor using a hollow‐fiber membrane was developed for the coupled transcription/translation system using T7 RNA polymerase and Escherichia coli S30 extract. The large surface area per the reaction volume of the reactor assured rapid mass transfers of substrates into the reaction mixture and of wastes out from it across the membrane by their molecular diffusion. The flux was large enough to maintain nucleotide concentrations for more than 3 h, which increased the protein synthesis greatly. In addition, the T7 terminator sequence downstream from the reporter genes was found to increase the synthesized protein significantly, especially when the product of polymerase chain reaction (PCR) was used as a template. Implementation of this finding and use of the bioreactor developed multiplied the productivity of protein by the in vitro direct expression from PCR template. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 194–199, 1999.