An easy cell-free protein synthesis system dependent on the addition of crude Escherichia coli tRNA.
The protein-synthesizing S30 extract of Escherichia coli contains tRNA, which limits its applications in cell-free protein synthesis. Here, we show that at least Arg- and Ser-acceptor activities can be removed from a standard S30 extract by treatment with an immobilized RNase A resin. This RNase-treated extract exhibits no protein synthesis activity, but regains it when supplied with crude E. coli tRNA and a small amount of human placental RNase inhibitor. The protein synthesis is dependent on the addition of tRNA in the presence of the RNase inhibitor. Chloramphenicol acetyltransferase was synthesized with this system and found to be active.
Kanda
T
T
Department of Industrial Chemistry, Chiba Institute of Technology, Tsudanuma, Narashino, Chiba 275-0016, Japan.
Takai
K
K
Yokoyama
S
S
Takaku
H
H
D001426:Bacterial Proteins / Q000096:biosynthesis*; D002474:Cell-Free System / Q000378:metabolism; D003636:DEAE-Cellulose; D004789:Enzyme Activation; D004800:Enzymes, Immobilized; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology; D012329:RNA, Bacterial / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D012116:Resins, Plant; D012259:Ribonuclease, Pancreatic; D012269:Ribosomal Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D012329:RNA, Bacterial; D012116:Resins, Plant; D012269:Ribosomal Proteins; C040469:ribosomal protein S30; D003636:DEAE-Cellulose; D012343:RNA, Transfer; D012259:Ribonuclease, Pancreatic