Purification And Characterization Of The Recombinant Bacillus Calmette-Gu E Rin Vaccine Ribonuclease ?

[Aims]The enzymatic cleavage of double-stranded(ds)RNA is essential for the maturation and decay of many eukaryotic and bacterial RNAs.Members of the ribonuclease ? family are primarily responsible for the cleavage of dsRNA and participate in dsRNA processing.The family can be classified into three classes.The ribonulease ? of Mycobacterium bovis BCG belongs to the first class which has the simplest structure with one nuclease domain and one double strands binding domain.In our study,we try to obtain an active recombinant Mycobacterium bovis BCG ribonulease ? with high purity for RNA cleavage assay by multiple steps of purification and partially described the biochemical characterization of this recombinant enzyme.[Methods]The gene encoding the ribonulease ? of Mycobacterium bovis BCG was cloned into pGEX-5X-1 and overexpressed in BL21(DE3)strain.The recombinant enzymes was extracted from host strain and purified with glutathione S-transferase(GST)affinity purification first.The preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS)was used to improve the purity of recombinant enzyme after the first affinity purification.After electroeluted from SDS gel block and electrodialysis,the 2-Methyl-2,4-pentanediol(MPD)was used to renature the recombinant enzyme denatured in the procedure of the preparative SDS-PAGE.The cleavage activity of the recombinant enzyme was tested with hairpin RNAs,BCG-16S[hp]RNA and BCG-23S[hp]RNA,designed on the stem region of the 16s rRNA presur and 23 s rRNA presur of Mycobacterium tuberculosis.And the RNA cleavage assay was performed with different types of metal ions to study the biochemical characterization of this recombinant enzymes.[Results]The expression of the full-length recombinant ribonulease III of Mycobacterium bovis BCG can be induced by IPTG in Escherichia coli BL21(DE3)strain.The purity of the full-length recombinant ribonulease III of Mycobacterium bovis BCG reached about 80%after the GST affinity purification,while it increased to nearly 99%through the purification with the preparative SDS-PAGE.The recombinant enzyme denatured in the procedure of the preparative SDS-PAGE did not show any activity in RNA cleavage activity but after renatured with 2-Methyl-2,4-pentanediol(MPD)and the further purification with a second GST affinity purification,the enzyme performed its activity and cleaved BCG-16S[hp]RNA into two fragments and BCG-23 S[hp]RNA into three fragments.The cleavage of RNA by the recombinant enzyme depended on special divalent ions and affected by the concentrations of divalent ions and monovalent ions.[Conclusions]The combination method of GST affinity purification and the preparative SDS-PAGE can be used to obtain the recombinant ribonulease ? of Mycobacterium bovis BCG with high purity.After renatured by an kind of amphipathic cosolvent,2-Methyl-2,4-pentanediol(MPD),the recombinant ribonuclease ? performed its activity in RNA cleavage.And the biochemical characterization of the recombinant ribonuclease ? performed with different metal ions in the following cleavage assay.It has not been discovered that the phenomenon and mechanism of RNA interfere by RNase ? in eukarytic cell existed in prokarytic cell.The regulation of mRNA in prokarytic cell by small RNA through RNase ? has been discovered.Our study provides the basic work for the further research of the characterization of RNase ? of Mycobacterium bovis BCG and its role in the small RNA metabolism in Mycobacterium bovis BCG.