Continuous-exchange cell-free protein synthesis using PCR-generated DNA and an RNase E-deficient extract.
Though the use of PCR-generated DNA (i.e., linear template) as template DNA is desirable because of its simple preparation, the linear template has not been routinely used in a conventional continuous-exchange cell-free (CECF) protein synthesis system due to the instability of the linear template and/or its transcript in the relatively long operation period. To overcome this problem and enhance soluble protein yield, an RNase E-deficient and molecular chaperone-enriched extract was used: (i) for compensating for the decrease in messenger RNA (mRNA) levels transcribed from the unstable linear template with improvement of mRNA stability by depletion of RNase E activity; and (ii) for enhancement of the soluble protein portion by assisting of the molecular chaperones. As a result, soluble erythropoietin production from a linear template was significantly enhanced in this modified CECF system using the RNase E-deficient and molecular chaperone-enriched extract, and the amount of soluble erythropoietin was estimated to be roughly 70% of that from a circular plasmid. We can conclude that the use of RNase E-deficient and molecular chaperone-enriched S30 extract mixture is effective in the enhancement of soluble protein expression from a linear template in the CECF system.
Jun
Soo Youn
SY
Chung-Ang University, Seoul, Korea.
Kang
Sang Hyeon
SH
Lee
Kwang-Ho
KH
D002474:Cell-Free System; D004269:DNA, Bacterial / Q000235:genetics*; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014176:Protein Biosynthesis / Q000502:physiology*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis*
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D004722:Endoribonucleases; C022537:ribonuclease E