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 x His-esat-6 fusion gene construction with histidine tag at its C-terminus. This recombinant plasmid was transformed into Escherichia coil 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 Ni2+-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). (C) 2013 Elsevier Inc. All rights reserved.