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 (MtEspI), is involved in repressing the activity of ESX-1-mediated secretion when the cellular ATP level is low. The ATP binding domain of MtEspI plays a crucial role in this regulatory process. However, further structural and functional studies of MtEspl are hindered due to the bottleneck of obtaining stable and pure recombinant protein. In this study, we systematically analyzed the structure and function of MtEspI using bioinformatics tools and tried various expression constructs to recombinantly express full-length and truncated MtEspI ATP binding domain. Finally, we prepared pure and stable MtEspl ATP binding domain, MtEspI(415-493), in Escherichia coli by fusion expression and purification with dual tag, Glutathione S-transferase (GST) tag and (His)(6) tag. P-31 NMR titration assay indicated that MtEspI(415-493) possessed a moderate affinity (similar to mu M) 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. (C) 2016 Elsevier Inc. All rights reserved.