分子伴侣共表达对核糖核酸酶R可溶性表达的影响

Effect of Co-expression of Molecular Chaperones on the Soluble Expression of Ribonuclease R

摘要:

核糖核酸外切酶R（RNase R）可降解几乎所有的线性RNA分子和Y结构RNA，但不易降解环形RNA（circ RNA）、套索结构（lariat RNA）和3’突出末端少于7个核苷酸的双链RNA分子，因此可以构建内含子cDNA文库用于可变剪切研究。本研究构建了含rnr基因的重组表达质粒pET-22b（+）-rnr，并将其表达产物纯化后通过SDS-PAGE分析和酶活性评价，确认rnr基因在大肠杆菌中已实现表达。之后构建4种分子伴侣共表达系统（pGro7、pKJE7、pTf16和pG-Tf2）。4种分子伴侣质粒分别与重组表达质粒pET-22b（+）-rnr共表达，筛选最适分子伴侣质粒，并优化共表达条件，提高目的蛋白可溶性表达。实验表明，分子伴侣质粒pGro7使蛋白表达量提高了43.80%，效果最为显著；20 ℃诱导时目的蛋白的可溶性表达最高；当L-阿拉伯糖浓度为0.50 mg/mL时，分子伴侣质粒pGro7使蛋白表达量提高了54.50%。通过使用分子伴侣共表达系统及优化表达条件提高了RNase R的可溶性表达，为该酶进一步研究奠定了基础。

Abstract:

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.