title: Cloning and expression of mycobacterial glutamine synthetase gene in Escherichia coli.
abstract: 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.
authors.affiliation: Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Jitendra
authors.initials: J
authors.lastname: Singh
authors.forename: Mohan Chandra
authors.initials: MC
authors.lastname: Joshi
authors.forename: Rakesh
authors.initials: R
authors.lastname: Bhatnagar
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005974:Glutamate-Ammonia Ligase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D005974:Glutamate-Ammonia Ligase