In the new era of “Omics”, the traditional techniques of protein expression in vivo can not come up with the exponential increase of genetic information. The cell-free protein synthesis system provides a new strategy of protein expression with advantages of rapid, convenient and high-throughput expression. The preparation of cell extracts, the optimization of substrate concentrations and the energy regeneration system are the key factors for the successful construction of cell-free protein expression system. In this work, the cell extract was prepared from RNase I- defective strain E. coli A19. The cell growth phase, the pressure for cell disruption and the storage condition of cell extracts were optimized. Meanwhile, the optimal substrate concentrations and the energy regeneration system were selected. Under the optimized conditions, the green fluorescent protein (GFP) reporter gene was expressed in the E. coli cell-free system with high expression level (Ca. 154 ?g/mL) which was 29 times higher than the expression level before optimization.
CHEN Haiqin, XU Zhinan, WANG Cheng, CEN Peilin, LIAO Yuhua
. Preliminary study on preparation of cell-free system for protein expression[J]. Frontiers of Chemical Science and Engineering, 2008
, 2(2)
: 224
-229
.
DOI: 10.1007/s11705-008-0029-9
1. Chen H Q Xu Z N Xu N Z Cen P L Efficient productionof a soluble fusion protein containing human beta-defensin-2 in E. coli cell-free systemJ Biotechnol 2005 115307315. doi:10.1016/j.jbiotec.2004.08.012
2. Xu Z N Chen H Q Yin X F Xu N Z Cen P L High-level expression of soluble humanβ-defensin-2 fused with green fluorescent protein in Escherichia coli cell-free systemAppl Biochem Biotech 2005 1275362. doi:10.1385/ABAB:127:1:053
3. Chen H Q Xu Z N Xu N Z Cen P L High levelexpression of human beta-defensin-2 gene with rare codons in E. coli cell-free systemProtein Peptide Lett 2006 13155161. doi:10.2174/092986606775101724
4. Rungpragayphan S Nakano H Yamane T PCR-linked in vitro expression: a novel system for high-throughputconstruction and screening of protein librariesFEBS Lett 2003 540147150. doi:10.1016/S0014‐5793(03)00251‐5
5. Busso D Kim R Kim S H Expression of soluble recombinant proteins in a cell-freesystem using a 96-well formatJ BiochemBioph Meth 2003 55233240. doi:10.1016/S0165‐022X(03)00049‐6
6. Sambrook J Fritsch E F Maniatis T Molecular cloning 3BeijingScience Press 2002 (In Chinese)
7. Hames B D Higgins S J Transcription and translation:a practical approachNew YorkIRL Press 1984 179209
8. Bradford M M Arapid and sensitive method for the quantitation of microgram quantitiesof protein utilizing the principle of protein-dye bindingAnal Biochem 1976 72248254. doi:10.1016/0003‐2697(76)90527‐3
9. Jewett M C Swartz J R Mimicking the Escherichia coli cytoplasmic environmentactivates long-lived and efficient cell-free protein synthesisBiotechnol Bioeng 2004 861926. doi:10.1002/bit.20026
