Efficient coupled transcription/translation from PCR template by a hollow-fiber membrane bioreactor.
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.
Nakano
H
H
Laboratory of Molecular Biotechnology, Department of Biological Mechanisms and Functions, Graduate School of Biological and Agricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Shinbata
T
T
Okumura
R
R
Sekiguchi
S
S
Fujishiro
M
M
Yamane
T
T
D001483:Base Sequence; D019149:Bioreactors; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D014176:Protein Biosynthesis; D013997:Time Factors; D014158:Transcription, Genetic; D014764:Viral Proteins
D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases