Kaur Gurkamaljit, Saini Varinder, Kumari Bandana, Kaur Jasbinder, Kaur Jagdeep
Department of Biotechnology, BMS Block-I, South Campus, Panjab University, Sector 25, Chandigarh, 160014, India.
Department of Pulmonary Medicine, Government Medical College and Hospital, Sec-32, Chandigarh, India.
Int J Biol Macromol. 2017 Aug;101:621-629. doi: 10.1016/j.ijbiomac.2017.03.096. Epub 2017 Mar 19.
Many mycobacterial proteins involved in lipid metabolism are reported to be essential for survival and pathogenesis of M. tuberculosis. Rv1076 of M. tuberculosis has been annotated as a putative esterase/lipase based on the consensus sequence 'GXSXG'. It is conserved in all the mycobacterial species. Therefore, in the present study we have characterized Rv1076 gene product in detail. The gene rv1076 was expressed in E. coli and purified from inclusion bodies with approx. 40% yield. The protein showed high specific activity with pNP- butyrate as preferred substrate. The enzyme was stable upto 50°C and in pH range of 6-8 i.e. under acidic conditions. Ser-140, Glu-239 and His-269 were confirmed as active site residues using site directed mutagenesis. The specific activity, K and V of enzyme was determined to be 177Umg protein, 334μM and 262μmolmlmin, respectively. Western blot analysis established Rv1076 to be an extracellular protein. Several putative immunodominant epitopes were predicted in Rv1076. Rv1076 elicited strong humoral response in both extrapulmonary and relapsed cases of TB patients. Therefore, we conclude that Rv1076 is a novel secretory esterase of M. tuberculosis which could be a potential immunodominant antigen of M. tuberculosis.
据报道，许多参与脂质代谢的分枝杆菌蛋白质对于结核分枝杆菌的生存和发病机制至关重要。基于共有序列“GXSXG”，结核分枝杆菌的Rv1076已被注释为推定的酯酶/脂肪酶。它在所有分枝杆菌物种中都保守。因此，在本研究中，我们详细表征了Rv1076基因产物。rv1076基因在大肠杆菌中表达，并从包涵体中纯化，产率约为40%。该蛋白以对硝基苯丁酸为首选底物时表现出高比活性。该酶在高达50°C的温度和pH值6 - 8的范围内即酸性条件下稳定。使用定点诱变确定Ser-140、Glu-239和His-269为活性位点残基。该酶的比活性、K和V分别测定为177Umg蛋白、334μM和262μmolmlmin。蛋白质印迹分析确定Rv1076是一种细胞外蛋白。在Rv1076中预测了几个推定的免疫显性表位。Rv1076在肺结核患者的肺外和复发病例中均引发强烈的体液反应。因此，我们得出结论，Rv1076是结核分枝杆菌的一种新型分泌性酯酶，可能是结核分枝杆菌的潜在免疫显性抗原。
