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

• The MtEspI ATP binding domain was solubly expressed in Escherichia coli.
• The purity of MtEspI415–493 was 96% after purified by GST- and His-dual-tag.
• Recombinant MtEspI415–493 possessed the ATP binding activity.

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 MtEspI 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 MtEspI ATP binding domain, MtEspI415–493, in Escherichia coli by fusion expression and purification with dual tag, Glutathione S-transferase (GST) tag and (His)6 tag. 31P NMR titration assay indicated that MtEspI415–493 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.