A continuous cell-free protein synthesis system for coupled transcription-translation.
A continuous cell-free protein synthesis system [Spirin et al. (1988) Science 242, 1162-1164] was modified so as to be suitable for coupled transcription-translation, a process useful for obtaining products of cloned genes or cDNAs. A reaction chamber equipped with an ultrafiltration membrane was newly designed and an HPLC pump was used to supply a low molecular weight substrate solution at a constant rate to the viscous reaction mixture in the chamber. By using an Escherichia coli S30 extract in this modified flow system (1 ml), coupled transcription-translation could be continuously performed for 17 h, the synthesized chloramphenicol acetyltransferase (congruent to 0.1 mg) being subsequently eluted through the chamber's membrane and then purified.
Kigawa
T
T
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo.
Yokoyama
S
S
D002474:Cell-Free System; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D014176:Protein Biosynthesis; D014158:Transcription, Genetic; D014462:Ultrafiltration
D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase