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

ABSTRACT

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.