Abstract
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.
Original language | English |
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Pages (from-to) | 61-69 |
Number of pages | 9 |
Journal | Journal of Biotechnology |
Volume | 34 |
Issue number | 1 |
DOIs | |
Publication status | Published - 1994 Apr 30 |
Externally published | Yes |
Keywords
- Dihydrofolate reductase
- E. coli extract
- Phosphorothioate
- Translation
- mRNA
ASJC Scopus subject areas
- Biotechnology
- Bioengineering
- Applied Microbiology and Biotechnology