Improvement of Escherichia coli cell-free system by utilization of cell extract having additional property. Problems and countermeasures.
In the Escherichia coli cell-free system, the modification of cell extract can be achieved by preparation of the strains carrying additional property or those being induced with a certain gene expression prior to harvesting. In this study, we analyzed the cell-free system with S30 extract containing T7 RNA polymerases (S30 extract-T7pol) prepared from E. coli BL21(DE3) strain, which includes T7 RNA polymerase from extrinsic genes by IPTG induction, as a model for the improvement of the cell-free system. The fact that a significant degree of mRNA degradation was observed in the cell-free system with S30 extract-T7pol indicates the increase of ribonuclease activity was an unfavorable influence derived from the cell-extract modification process. We also showed that this influence was settled by the addition of an effective ribonuclease inhibitor, such as copper (II) ion, to the reaction mixture.
Nishimura
N
N
Biotechnology Research Laboratory, Kobe Steel Ltd., Tsukuba, Japan.
Kitaoka
Y
Y
Niwano
M
M
D003300:Copper / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins
D012333:RNA, Messenger; D014764:Viral Proteins; D003300:Copper; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases