Singh Jitendra, Joshi Mohan Chandra, Bhatnagar Rakesh
Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.
Biochem Biophys Res Commun. 2004 Apr 30;317(2):634-8. doi: 10.1016/j.bbrc.2004.03.094.
Extracellular glutamine synthetase (GS) is one of the prominent proteins secreted by pathogenic mycobacteria such as Mycobacterium tuberculosis and Mycobacterium bovis. Non-pathogenic species like Mycobacterium smegmatis and Mycobacterium phlei do not secrete this protein. GS has been proposed to play an indispensable role in intracellular survival of pathogenic mycobacteria. In this study, the structural gene for extracellular glutamine synthetase of M. tuberculosis was PCR amplified and expressed as fusion protein with hexahistidine residues in Escherichia coli. Expression of GS in E. coli under transcriptional regulation of T5 promoter yielded an insoluble protein aggregating to form inclusion bodies. The inclusion bodies were solubilized in presence of 8 M urea and the enzyme was purified to homogeneity under denaturing conditions using nitrilotriacetic acid (Ni-NTA) affinity chromatography. The denatured protein was renatured by gradual removal of the urea while immobilized on (Ni-NTA) column. The yield of purified recombinant glutamine synthetase was 40 mg/L. The purified recombinant enzyme was obtained in highly active state having specific activity of 200 U/mg protein. This is the first report describing cloning and expression of mycobacterial glutamine synthetase gene in E. coli.
细胞外谷氨酰胺合成酶（GS）是结核分枝杆菌和牛分枝杆菌等致病性分枝杆菌分泌的主要蛋白质之一。耻垢分枝杆菌和草分枝杆菌等非致病性分枝杆菌不分泌这种蛋白质。有人提出GS在致病性分枝杆菌的细胞内存活中起不可或缺的作用。在本研究中，结核分枝杆菌细胞外谷氨酰胺合成酶的结构基因通过PCR扩增，并在大肠杆菌中作为带有六个组氨酸残基的融合蛋白表达。在T5启动子的转录调控下，GS在大肠杆菌中的表达产生了一种不溶性蛋白质，聚集形成包涵体。包涵体在8 M尿素存在下溶解，然后使用次氮基三乙酸（Ni-NTA）亲和色谱在变性条件下将酶纯化至同质。当固定在（Ni-NTA）柱上时，通过逐渐去除尿素使变性蛋白复性。纯化的重组谷氨酰胺合成酶的产量为40 mg/L。纯化的重组酶以高活性状态获得，比活性为200 U/mg蛋白质。这是第一篇描述分枝杆菌谷氨酰胺合成酶基因在大肠杆菌中克隆和表达的报告。
