关键词: 结核分枝杆菌H37Rv, Rv0357c蛋白, 蛋白异源表达, 亲和层析, 生物信息学

Abstract: The aim of this work was to reveal the biological function of Rv0357c from Mycobacterium tuberculosis (Mtb) for the further exploration of a novel anti-Mtb drug target. The bioinformatic analyses of Rv0357c were conducted; the prokaryotic expression vector pET-Rv0357c for expressing Rv0357c fusion protein was subsequently constructed and introduced into Escherichia coli BL21(DE3) competent cells followed by over-production of the Rv0357c fusion protein; Rv0357c fusion protein was purified by the immobilized cobalt affinity chromatography and subjected to protein identification together with enzymatic assay. The bioinformatic analyses revealed that Rv0357c was an acidic stable hydrophilic protein containing two conserved motifs and conserved active site residues observed in previously identified adenylosuccinate synthetases (AdSSs), and it showed low sequence identities(＜40%) to human enzymes (muscle isoform, AdSS1, and non-muscle isoform, AdSS2). Based on the SDS-PAGE analysis, the Rv0357c fusion protein was found to be expressed as inclusion bodies with an apparent subunit molecular mass of 48 ku, and the target enzyme in soluble form could be obtained after solubilizing the inclusion bodies, affinity chromatography and subsequent protein refolding with a refolding yield of approximate 63%. The overproduced protein obtained here was further identified as the target protein as demonstrated by Western Blot analysis using Anti-6×His antibody. Furthermore, enzymatic assay revealed that the biological activity of AdSS could be detected using refolded Rv0357c fusion protein with a specific activity of 0.016 1 U/mg. In summary, the Rv0357c is a functional enzyme with AdSS activity. This work will lay an experimental foundation for the further functional identification and the development of a new drug target against Mtb.

Key words: Mycobacterium tuberculosis H37Rv, Rv0357c, heterologous expression, affinity chromatography, bioinformatics