Recombinant preparation and functional studies of EspI ATP binding domain from Mycobacterium tuberculosis.

Abstract The ESX-1 secretion system of Mycobacterium tuberculosis is required for the virulence of tubercle bacillus. EspI, the ESX-1 secretion-associated protein in Mycobacterium tuberculosis ( Mt EspI), is involved in repressing the activity of ESX-1-mediated secretion when the cellular ATP level is low. The ATP binding domain of Mt EspI plays a crucial role in this regulatory process. However, further structural and functional studies of Mt EspI are hindered due to the bottleneck of obtaining stable and pure recombinant protein. In this study, we systematically analyzed the structure and function of Mt EspI using bioinformatics tools and tried various expression constructs to recombinantly express full-length and truncated Mt EspI ATP binding domain. Finally, we prepared pure and stable Mt EspI ATP binding domain, Mt EspI 415–493 , in Escherichia coli by fusion expression and purification with dual tag, Glutathione S-transferase (GST) tag and (His) 6 tag. 31 P NMR titration assay indicated that Mt EspI 415–493 possessed a moderate affinity (∼μM) for ATP and the residue K425 was located at the binding site. The protocol described here may provide a train of thought for recombinant preparation of other ESX-1 secretion-associated proteins.