科研之友
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摘要
Ribonucleic acid exonuclease R (RNase R) can degrade almost all the linear RNA molecules and Y-structured RNAs, but does not readily degrade circRNAs, lariat RNAs and double-stranded RNA molecules with less than 7 nucleosides Therefore, intron cDNA library can be constructed for the intron splicing variable research. In this study, a recombinant expression plasmid pET-22b(+) -rnr containing RNR gene was constructed. The expression product was purified and analyzed by SDS-PAGE while the enzyme activity was also determined to confirm that the rnr gene has been expressed in E. coli. Then, four molecular chaperone co-expression systems (pGro7, pKJE7, pTf16 and pG-Tf2) were constructed. The four molecular chaperone plasmids were co-expressed respectively with the recombinant expression plasmid pET-22b(+)-rnr. The optimal molecular chaperone plasmid was screened and the co-expression conditions were optimized to improve the soluble expression of the target protein. The results showed that the molecular chaperone plasmid pGro7 was the most effective and could increase the expression of protein by 43.80%, with the soluble expression of the target protein reaching the highest at an inducing temperature of 20 ℃. When the concentration of L-arabinose was 0.50 mg/mL, the molecular chaperone plasmid pGro7 increased the expression of protein by 54.50%. The soluble expression of RNase R could be improved by using the molecular chaperone co-expression system and optimizing the expression conditions, which laid a foundation for further study of RNase R.
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