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

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. (31)P 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.

Expression, purification and characterization of Esx-1 secretion-associated protein EspL from Mycobacterium tuberculosis.

The Esx-1 cluster encodes a special secretion system that is important for granuloma formation and virulence when Mycobacterium tuberculosis infects the host. As one of the 'core' genes in the cluster, Rv3880c gene codes an Esx-1 secretion-associated protein EspL from Mycobacterium tuberculosis (MtEspL). It has been reported that EspL had a strong influence on the secretion of other two virulence factors, EsxA and EspE. However, so far little is known about the tertiary structure and specific function of MtEspL due to the difficulty in preparing the high-quality protein. In this study, we tried several fusion tags and various expression conditions to recombinantly express MtEspL. Through a four-step purification procedure, ultimately, we successfully prepared the full-length MtEspL in Escherichia coli BL21 (DE3) with a purity of 98%. The yields of the purified MtEspL protein were 14 mg/L in Luria Bertani medium and 5.6 mg/L in M9 minimal medium, respectively. Biophysical experiments showed that MtEspL existed in a dimeric form. Moreover, the (1)H-(15)N HSQC spectrum recorded on MtEspL illustrates a favorable dispersion of the resonance peaks, indicating that the symmetric dimeric MtEspL adopted a well-folded structure and might be feasible to determine its solution structure by NMR spectroscopy. Moreover, we identified a strong DNA-binding ability of MtEspL with fluorescence quenching experiments. Our work lays the basis for further structural determination and functional exploration of MtEspL.