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

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 1H-15N 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.
Source: Protein Expression and Purification - Category: Biochemistry Source Type: research