Cloning, expression and N-terminal formylation of ESAT-6 of Mycobacterium tuberculosis H37Rv.
Bacterial protein synthesis initiates with a formyl-methionine group. Addition of the antibiotic actinonin, a known peptide deformylase inhibitor, at the time of induction of protein expression results in the retention of the formyl group by the overexpressed protein. We have demonstrated the application of this system to obtain N-formylated form of a mycobacterial secretory protein ESAT-6 which is an important target for T-cell recognition during Mycobacterium tuberculosis infection. The gene encoding the ESAT-6 of M. tuberculosis was inserted into a bacterial expression vector pET23a (+) resulting in a 6 × His-esat-6 fusion gene construction with histidine tag at its C-terminus. This recombinant plasmid was transformed into Escherichia coli strain BL21(DE3) and effectively expressed in presence or absence of a peptide deformylase inhibitor, actinonin. The expressed fusion proteins (non-formylated and N-formylated ESAT-6) found in the inclusion bodies were purified by Ni(2+)-NTA chromatography. The N-terminal formylation of ESAT-6 was confirmed by advanced proteomic techniques including MALDI-TOF mass spectrometry and 2-DE. The gene encoding ESAT-6 of M. tuberculosis was successfully cloned and expressed in E. coli. The N-terminal formylation of this protein (ESAT-6) was carried out by inducing the recombinant clone harboring the ESAT-6 gene in presence of actinonin (peptide deformylase inhibitor).
Department of Biochemistry, Postgraduate Institute of Medical Education & Research, Chandigarh 160012, India.
Shabir Ahmad
SA
Mir
Sadhna
S
Sharma
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D001669:Biochemical Phenomena; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D006639:Histidine; D006877:Hydroxamic Acids; D051379:Mice; D008807:Mice, Inbred BALB C; D009842:Oligopeptides; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D032581:Tuberculosis Vaccines
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; C471213:His-His-His-His-His-His; D006877:Hydroxamic Acids; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D032581:Tuberculosis Vaccines; D006639:Histidine; C010531:actinonin
2-DE and mass spectrometry; Actinonin; Antigen; Isoelectric form