结核分枝杆菌Rv0357c蛋白的生物信息学分析与鉴定

Bioinformatic analysis and identification of Rv0357c from Mycobacterium tuberculosis

为鉴定结核分枝杆菌(Mycobacterium tuberculosis,Mtb)Rv0357c蛋白的生物学功能,进而寻找新的抗Mtb靶点,对Rv0357c进行生物信息学分析,构建了表达Rv0357c的原核表达载体pET-Rv0357c,并在大肠杆菌BL21(DE3)菌株中表达Rv0357c融合蛋白,最后用Co 2+亲和层析纯化并对其进行鉴定与活性分析。生物信息学分析表明:Rv0357c是稳定的酸性亲水蛋白,且含有腺苷酸琥珀酸合成酶(AdSS)的2个保守基序和保守的活性位点,同时和人AdSS同工酶(AdSS1和AdSS2)的同源性均低于40%。SDS-PAGE分析证实Rv0357c融合蛋白以包涵体形式表达,亚基分子质量约为48 ku,经包涵体溶解、亲和纯化和重折叠,可以得到可溶性目的蛋白(复性率约为63%)。Western Blot检测进一步表明过表达的蛋白为目的蛋白。此外,酶活性测定证实重折叠的Rv0357c融合蛋白有AdSS活性,比活力为0.0161 U/mg。综上,Rv0357c是功能性的AdSS蛋白,这将为Rv0357c的进一步功能鉴定和开发新型抗Mtb靶点奠定基础。

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)was less than 40%.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 0.0161 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.