Removing tRNA from a cell-free protein synthesis system for use in protein production.
The cell-free system for biosynthesis of proteins is becoming an important tool for protein engineering. In particular, introduction of the unnatural amino acids is achieved though cell-free protein synthesis with the use of chemically acylated tRNA that recognizes a specific codon. In the original method, however, it was difficult to control the system through changing tRNA composition, as the endogenous tRNAs are involved in the reaction. Thus, in the present study, we digested the tRNA within Escherichia coli S30 extract with resin-bound RNase A, and estimated the protein synthesis activity. It was revealed that this digestion process does not damage the activity, if a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), is present in the digestion reaction.
Kanda
T
T
Department of Industrial Chemistry, Chiba Institute of Technology, Japan.
Takai
K
K
Yokoyama
S
S
Takaku
H
H
D002474:Cell-Free System; D004926:Escherichia coli / Q000378:metabolism*; D007202:Indicators and Reagents; D010664:Phenylmethylsulfonyl Fluoride; D011480:Protease Inhibitors; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D012343:RNA, Transfer / Q000302:isolation & purification* / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic
D007202:Indicators and Reagents; D011480:Protease Inhibitors; D010664:Phenylmethylsulfonyl Fluoride; D012343:RNA, Transfer; D012259:Ribonuclease, Pancreatic