Expression And Optimization Of RNase R In Escherichia Coli

In recent years,RNA research,especially circular RNA,is one of the emerging hotspots in the field of biomedical research.The activity of RNase R directly affects the quality of circular RNA after treatment.Therefore,the production and purification of RNase R is worthy of further investigation.Exonuclease R(RNase R)degrades almost all linear RNA molecules and Y-structured RNA,but it does not readily degrades circular RNA(circRNA),lassot RNA,and double-stranded RNA molecules with less than 7 nucleosides acid at 3' overhangs,and intron cDNA libraries can be constructed for variable splicing studies.In this study,the ribonuclease R derived from Escherichia coli was found from the NCBI gene bank,and the strain E.coli K12 containing the target gene was purchased from GDMCC.Then the genome was extracted and used as a template for PCR to obtain the definite gene.The main contents and results of this study are as follows:After the target gene rnr amplified by PCR,the recombinant plasmid pET22b-rnr was constructed and transferred into E.coli BL21(DE3),and then the recombinant Escherichia coli BL21(DE3)/pET22b-rnr was selected.The recombinant strain was induced in LB medium to express the enzyme protein,and its molecular weight was analyzed by SDS-PAGE.Purified recombinant RNase R was obtained by affinity chromatography on Ni column,and the enzyme activity was determined by using S.cerevisiae Total RNA as substrate.The comparison with RNase R(GESENEED)showed that the activity of RNase R prepared by this experiment was close to or more than commercial enzymes.Because of the difference in the expression of E.coli exogenous proteins under different conditions,this study used a single factor optimization experiment to induce expression conditions of recombinant E.coli BL21(DE3)/ pET22b-rnr which had successfully expressed RNase R.The experimental results show that the optimal induction temperature of BL21(DE3)/ pET22b-rnr is 20 °C,the optimal induction point(OD600)is 0.8,the optimal IPTG induction dose is 0.8 mM,and the optimal induction time is 4h.At this time,the soluble expression level of the target protein was the highest,reaching 79.26 mg/L,which was 54.7% higher than the starting condition.In this study,four molecular chaperone co-expression systems(four chaperone plasmids,which were pGro7,pKJE7,pTf16 and pG-Tf2,were co-expressed with the recombinant expression plasmid pET22b-rnr)were constructed,and the optimal molecular chaperone plasmid was screened.After that we optimize co-expression conditions to improve the soluble expression of the target protein.The results showed that the molecular chaperone plasmid pGro7 increased the protein expression by 43.80%,and the effect was the most significant.The soluble protein of the target protein was the highest when induced at 20 °C.When the Larabinose concentration was 0.50 mg/mL,the amount of expression increased to 82.98 mg/L.The soluble expression of RNase R was improved by using the molecular chaperone coexpression system and optimizing the expression conditions,which laid a foundation for further study of the enzyme.