Efficient expression of E. coli dihydrofolate reductase gene by an in vitro translation system using phosphorothioate...

Dihydrofolate reductase (DHFR) of Escherichia coli (E. coli) was synthesized in a cell-free translation system of E. coli directed by phosphorothioate-containing mRNA (thio-mRNA) which was polymerized by an in vitro transcription of the DHFR gene in the presence of SP diastereomers of ribonucleoside 5'-O-(1-thiotriphosphates). The molecular weights of the products thus obtained were identical to those with the unsubstituted mRNA. Furthermore, the thio-mRNA for DHFR showed higher translational activities than the corresponding unsubstituted mRNA. It is suggested that this effectiveness resulted from the higher stability of thio-mRNA in the cell-free translation system. Amongst the various types of thio-mRNAs, the single substitution of adenosine residues was most effective in translational activity. This higher translational activity of thio-mRNA compared with the unsubstituted mRNA was also demonstrated in a continuous flow cell-free system originally developed by Spirin et al. (1988). Therefore, introduction of sulfur atoms into phosphodiester bonds of mRNA appears to be a useful strategy for the stabilization of mRNA in large-scale protein production in vitro.