摘要
目的:探索结核杆菌异柠檬酸裂合酶(ICL)蛋白322位点赖氨酸(Lys322)的乙酰化修饰对蛋白功能的调控作用。方法:构建结核杆菌ICL蛋白原核表达载体p ET28a-icl,并对Lys322位点进行定点突变为精氨酸(Arg,R)和谷氨酰胺(Glu,Q),体外表达纯化获得重组蛋白ICLWT、ICL322R和ICL322Q。通过Western blotting和酶活性测定来揭示Lys322位点突变前后对蛋白的乙酰化修饰水平及蛋白功能的影响。结果:Western blotting检测发现大肠杆菌表达体系获得的ICLWT、ICL322R和ICL322Q蛋白均有较高水平的蛋白赖氨酸乙酰化修饰信号,较ICLWT,ICL322R和ICL322Q突变蛋白的酶活性分别下降了大约50%和70%。结论:在大肠杆菌的表达体系中,ICL蛋白可以获得乙酰化修饰。ICL322Q突变蛋白酶活性的显著下降,揭示Lys322位点乙酰化修饰对ICL蛋白的功能存在负向调控。为未来深入探索赖氨酸乙酰化修饰对结核杆菌代谢,潜伏感染的调控作用奠定了基础。
Objective： Isocitrate lyase（ ICL） of Mycobacterium tuberculosis（ Mtb） was acetylated at lysine322. The aim was to investigate ICL Lys322 acetylation and its regulatory function in activity of ICL. Methods：The prokaryotic expression plasmids pET28a-icl was constructed and Lys322 of ICL was site-directed mutated into arginine（ Arg,R） and glutamine（ Gln,Q）,respectively. Then recombinant proteins ICLWT,ICL322 Rand ICL322Q were expressed and purified in vitro. Acetylation of wild type ICL and its mutants were examined by Western blotting with anti-acetyllysine antibody,and enzyme activities of proteins were evaluated. Results： Western blotting showed that ICLWT,ICL322 Rand ICL322Qwere indeed acetylated proteins. Compared with activity of ICLWT,mutation of Lys322 to arginine decreased the enzyme activity by approximately 50%,and glutamine substitution dramatically decreased the activity more than 70%. Conclusion： The results of Western blotting showed that the ICL and two mutant proteins can be acetylated effectively in E. coli expression system. Decreasing in ICL322 Q activity suggested that Lys322 acetylation could negatively regulate isocitrate lyase of Mycobacterium tuberculosis.This provided a foundation to study an important aspect of Mtb,the metabolic regulation of Mtb,which may lead to new knowledge on latent infection.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2015年第6期8-13,共6页
China Biotechnology
基金
国家自然科学基金资助项目(81261120558
30901828)
