Biochemical and Biophysical Research Communications, Vol.338, No.3, 1346-1352, 2005
Cell-free synthesis of recombinant proteins from PCR-amplified genes at a comparable productivity to that of plasmid-based reactions
The functional stability of mRNA is one of the Crucial factors affecting the efficiency of cell-free protein synthesis. The importance of the stability of mRNA in the prolonged synthesis of protein molecules becomes even greater when the cell-free protein synthesis is directed by PCR-amplified DNAs, because the linear DNAs are rapidly degraded by the endogenous nucleases and, thus, the continuous generation of mRNA molecules is limited. With the aim of developing a highly efficient cell-free protein synthesis system directed by PCR products, in this study, we describe a systematic approach to enhance the stability of mRNA in cell-free extracts. First, exonuclease-mediated degradation was substantially reduced by introducing a stem-loop structure at the 3'end of the mRNA. The endonucleolytic cleavage of the mRNA was minimized by using an S30 extract prepared from an Escherichia coli strain that is deficient in a major endonuclease (RNase E). Taken together, through the retardation of the endonucleolytic and exonucleolytic degradations of the mRNA molecules, the level of protein expression from the PCR-amplified DNA templates becomes comparable to that of conventional plasmid-based reactions. The enhanced productivity Of the PCR-based cell-free protein synthesis enables the high-throughput generation of protein molecules required for many post-genomic applications. (c) 2005 Elsevier Inc. All rights reserved.
Keywords:mRNA stability;T7 terminator;endonuclease;exonuclease;PCR;cell-free protein synthesis;rne 131;high-throughput protein expression