EFFICIENT EXPRESSION OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE GENEBY AN IN-VITRO TRANSLATION SYSTEM USING PHOSPHOROTHIOATE MESSENGER-RNA

Dihydrofolate reductase (DHFR) of Escherichia call (E. coli) was synth esized in a cell-free translation system of E. coli directed by phosph orothioate-containing mRNA (thio-mRNA) which was polymerized by an in vitro transcription of the DHFR gene in the presence of S-P diastereom ers of ribonucleoside 5'-O-(1-thiotriphosphates). The molecular weight s of the products thus obtained were identical to those with the unsub stituted mRNA. Furthermore, the thio-mRNA for DHFR showed higher trans lational activities than the corresponding unsubstituted mRNA. It is s uggested that this effectiveness resulted from the higher stability of thio-mRNA in the cell-free translation system. Amongst the various ty pes of thio-mRNAs, the single substitution of adenosine residues was m ost effective in translational activity. This higher translational act ivity of thio-mRNA compared with the unsubstituted mRNA was also demon strated in a continuous flow cell-free system originally developed by Spirin et al. (1988). Therefore, introduction of sulfur atoms into pho sphodiester bonds of mRNA appears to be a useful strategy for the stab ilization of mRNA in large-scale protein production in vitro.