Journal
BIOCHEMICAL ENGINEERING JOURNAL
Volume 138, Issue -, Pages 47-53Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bej.2018.07.001
Keywords
In vitro protein synthesis; Purified components; Cell-free system; Titration
Funding
- Ministry of Science and Technology of China 973 Grants [2012CB721000, 2011CBA00800]
- 863 Grants [2012AA02A701]
- National Natural Science Foundation of China [31222002, 81370872]
- Science and Technology Department of Hubei Province
- J1 Biotech Co. Ltd. [2014091610010595]
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Cell-free protein synthesis (CFPS) is a valuable tool for basic and applied research. The Escherichia coli cell lysate-based system is the most widely used for in vitro transcription and translation (TX-TL). To optimize this system and increase target protein production, the present study investigated the contribution of most of the macromolecular components of the TX-TL machinery by means of systematic titration experiments. A 1.9-fold increase in enhanced green fluorescent protein expression was achieved relative to the reference system by single-component supplementation, while a 2.5-fold increase was achieved by adding T7 RNA polymerase, ribosome-recycling factor, translation initial factor (IF) 1, IF2, and elongation factor Tu. A similar enhancement in the production of other proteins was also achieved by supplementation, thereby indicating the broad applicability of this approach. These results provide useful information for further optimization of CFPS and a basis for improving industrial enzyme production by engineering bacterial strains that express specific components of the TX-TL machinery. (C) 2018 Elsevier B.V. All rights reserved.
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