Hao Fang, Pei Xiu-ying, Zhang Xue-lian, Zhang Shun-bao, Lai Xu-hui, Wang Hong-hai
Institute of Genetic, Fudan University, Shanghai 200433, China.
Zhonghua Jie He He Hu Xi Za Zhi. 2005 Dec;28(12):845-8.
To obtain recombinant protein with enzymatic activities of isocitrate lyase (ICL).
The icl gene was amplified by polymerase chain reaction (PCR) from Mycobacterium tuberculosis H(37)Rv strain genomic DNA and cloned into pET28-a(+) vector. The recombinant protein was expressed in E.coli BL21 (DE3). Enzyme activity of the protein was assayed after purifying with Ni-NTA resin.
The recombinant ICL was purified in a highly active state with a specific activity of about 7.657 x 10(2) micromol x mg(-1) x min(-1). The pH curve indicated that recombinant ICL activity was optimal at pH 7.4. The LC/MS spectrometry showed a 50 603.347 molecular mass of recombinant ICL. The CD spectrum showed that the percentages for alpha- helix, beta- sheet, beta- turn, and random coil were 43.8%, 31.9%, 3.4%, and 20.9%, respectively.
The icl gene of Mycobacterium tuberculosis H(37)Rv was successfully cloned and expressed. The enzymatic properties demonstrated the purified recombinant protein had activities of ICL. This work can facilitate immunologic research and the discovery of novel antimicrobial agents against Mycobacterium tuberculosis.
获得具有异柠檬酸裂解酶（ICL）酶活性的重组蛋白。
通过聚合酶链反应（PCR）从结核分枝杆菌H（37）Rv菌株基因组DNA中扩增icl基因，并克隆到pET28-a（+）载体中。重组蛋白在大肠杆菌BL21（DE3）中表达。用Ni-NTA树脂纯化后测定该蛋白的酶活性。
重组ICL以高活性状态纯化，比活性约为7.657×10（2）微摩尔×毫克（-1）×分钟（-1）。pH曲线表明重组ICL活性在pH 7.4时最佳。LC/MS光谱显示重组ICL的分子量为50 603.347。圆二色光谱显示α-螺旋、β-折叠、β-转角和无规卷曲的百分比分别为43.8%、31.9%、3.4%和20.9%。
结核分枝杆菌H（37）Rv的icl基因成功克隆并表达。酶学性质表明纯化的重组蛋白具有ICL活性。这项工作有助于免疫研究以及发现针对结核分枝杆菌的新型抗菌剂。
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