Journal
BIOTECHNIQUES
Volume 44, Issue 3, Pages 387-391Publisher
FUTURE SCI LTD
DOI: 10.2144/000112690
Keywords
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Funding
- Korea Science and Engineering Foundation [R01-2004-000-10375-0]
- National Research Foundation of Korea [R01-2004-000-10375-0] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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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 enhancenment of soluble protein expression from a linear template in the CECF system.
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