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 coup led transcription/translation system using T7 RNA polymerase and Escherichi a coli S30 extract. The large surface area per the reaction volume of the r eactor 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 T 7 terminator sequence downstream from the reporter genes was found to incre ase the synthesized protein significantly, especially when the product of p olymerase chain reaction (PCR) was used as a template. Implementation of th is finding and use of the bioreactor developed multiplied the productivity of protein by the in vitro direct expression from PCR template. (C) 1999 Jo hn Wiley & Sons, Inc.