Fig. 2: Purification and characterization of LhgR.

a SDS-PAGE analysis of the purification of LhgR. Lane M, molecular weight markers; lane 1, crude extract of E. coli BL21(DE3) harboring pETDuet-lhgR; lane 2, purified His₆-tagged LhgR using a HisTrap column. b Gel-filtration chromatography of the purified LhgR with the Superdex 200 10/300 GL column. Red curve, the chromatogram of purified LhgR; Black line, a standard curve for protein molecular mass standards. c LhgR can bind to the lhgO promoter region. F1 fragment containing the lhgO promoter region (10 nM) was titrated by purified LhgR (0, 20, 40, 60, 80, 100, 120, 140, 160 nM). Lane M, molecular weight markers. d DNase I footprinting analysis of LhgR binding to the lhgO promoter region. The F1 fragment was labeled with 6-carboxyfluorescein (FAM) and incubated with 2 μg LhgR (blue line) or without LhgR (red line). The region protected by LhgR is indicated with a dotted box. e l-2-HG prevents LhgR binding to the lhgO promoter region. EMSAs were carried out with F1 fragment (10 nM) and purified LhgR (60 nM) in the absence of any other tested compounds (No ligand) and in the presence of 50 mM different compounds. Lane M was the molecular weight marker; lane 0 without LhgR was used as the control.