Expression And Identification Of M. Tuberculosis Rv1096

The cell wall of Mycobacterium tuberculosis plays an important role in infecting host and being survival for long time in the host. The cell wall core of Mycobacterium tuberculosis is composed of mycolic acid arabinogalactan and peptidoglycan. Peptidoglycan is a net-like macromolecule formed from linear glycan chains, linked by short peptides. The glycan chains are builted up by alternating units of N-acetyl-glucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) linked byβ-1, 4 glycoside bonds. Peptidoglycan is responsible for maintining bacterial cell shape. There are some proteins and enzymes associated with the cell wall of M. tuberculosis. To characterize these proteins and enzymes is critical to understanding infection mechanism of M. tuberculosis to host, bacterial survival and immune modulation in the host as well as synthesis and degradation of cell wall macromolecules.M. tuberculosis Rv1096 is a cell wall protein. The bioinformatic analysis of Rv1096 showed that Rv1096 has 31% (76/243) identity with peptidoglycan GlcNAc deacetylase (PgdA) of Streptococcus pneumoniae. The deacetylation of peptidoglycan in S. pneumoniae causes significantly increase bacterial resistance to lysozyme hydrolysis and survival in host. We will identify the function of Rv1096 in this study.The objectives of this study: (1) To amplify Rv1096 gene from M. tuberculosis H37Rv genome by high fidelity DNA polymerase and clone it to pMD18-T vector. (2) To construct pET29b-Rv1096 express plasmid and to express soluble Rv1096 protein in E. coli BL21(DE3)pLysS. (3) To purify Rv1096 protein by affinity chromatography and confirm the purified protein by SDS-PAGE and Western blotting. (4) To identify the fuction of Rv1096 protein.The results are followings:1. M. tuberculosis Rv1096 was cloned.Rv1096 gene was amplified by PrimeStar DNA polymerase from Mycobacterium tuberculosis H37Rv genome and ligated to pMD18-T vector to generate pMD18-Rv1096. pMD18-Rv1096 was confirmed by NdeI digestion followed by DNA sequencing.2. Expression plasmid pET29b-Rv1096 was constructed.Rv1096 was subcloned to pET29b plasmid to yield an expression plasmid pET29b-Rv1096. pET29b-Rv1096 was transformed to E. coli BL21(DE3)pLysS.3. Soluble Rv1096 protein was produced in E. coli BL21(DE3)pLysS.Expression of Rv1096 protein was induced with 2.5 mM IPTG at 18°C for 12 hours. After sonication, both supernatant and pellet fractions were analyzed by Western blotting. The Rv1096 protein with expected 31.07 kD is soluble.4. Rv1096 protein was purified by affinity chromatography.The C-terminus of Rv1096 was fused with histidine tag in pET29b. Therefore, Rv1096 was purified by histidine-Ni2+ affinity chromatography. The Rv1096 protein in elution fractions 1, 2 were confirmed by Western blotting. The elution fraction 1 (53.78μg/ml) was used for enzyme assay.5. The enzyme assay of peptidoglycan GlcNAc deacetylase was set up.The purified Rv1096 protein was incubated with M. smegmatis peptidoglycan as a substrate at 37℃for 3 h. The acetyl group was detected by using Acetic Acid Detection Kit. If Rv1096 had deacetylase activity the acetyl group was released from the reaction and converted to acetic acid which was detected by measuring OD of NADH+H+ at 340 nm after a serial reactions. The results showed that Rv1096 protein has peptidoglycan deacetylase activity.Conclusions:In this study, we cloned Rv1096 gene and constructed pET29b-Rv1096 expression vector. We acquired soluble Rv1096 protein in E. coli BL21(DE3)pLysS and identified the purified Rv1096 protein as peptidoglycan deacetylase.