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 |
|---|---|
| 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 |
Fingerprint
Keywords
- Dihydrofolate reductase
- E. coli extract
- mRNA
- Phosphorothioate
- Translation
ASJC Scopus subject areas
- Biotechnology
- Applied Microbiology and Biotechnology
Cite this
Efficient expression of E. coli dihydrofolate reductase gene by an in vitro translation system using phosphorothioate mRNA. / Tohda, Hideki; Chikazumi, Nobutoshi; Ueda, Takuya; Nishikawa, Kazuya; Watanabe, Kimitsuna.
In: Journal of Biotechnology, Vol. 34, No. 1, 30.04.1994, p. 61-69.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Efficient expression of E. coli dihydrofolate reductase gene by an in vitro translation system using phosphorothioate mRNA
AU - Tohda, Hideki
AU - Chikazumi, Nobutoshi
AU - Ueda, Takuya
AU - Nishikawa, Kazuya
AU - Watanabe, Kimitsuna
PY - 1994/4/30
Y1 - 1994/4/30
N2 - 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.
AB - 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.
KW - Dihydrofolate reductase
KW - E. coli extract
KW - mRNA
KW - Phosphorothioate
KW - Translation
UR - http://www.scopus.com/inward/record.url?scp=0028280813&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0028280813&partnerID=8YFLogxK
U2 - 10.1016/0168-1656(94)90166-X
DO - 10.1016/0168-1656(94)90166-X
M3 - Article
C2 - 7764744
AN - SCOPUS:0028280813
VL - 34
SP - 61
EP - 69
JO - Journal of Biotechnology
JF - Journal of Biotechnology
SN - 0168-1656
IS - 1
ER -