Continuous-exchange cell-free protein synthesis using PCR-generated DNA and an RNase E-deficient extract
- Authors
- Jun, Soo Youn; Kang, Sang Hyeon; Lee, Kwang-Ho
- Issue Date
- Mar-2008
- Publisher
- FUTURE SCI LTD
- Citation
- BIOTECHNIQUES, v.44, no.3, pp 387 - 391
- Pages
- 5
- Journal Title
- BIOTECHNIQUES
- Volume
- 44
- Number
- 3
- Start Page
- 387
- End Page
- 391
- URI
- https://scholarworks.bwise.kr/cau/handle/2019.sw.cau/23835
- DOI
- 10.2144/000112690
- ISSN
- 0736-6205
1940-9818
- Abstract
- 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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Collections - College of Natural Sciences > Department of Life Science > 1. Journal Articles
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