Gel filtration chromatography.

To estimate the relative molecular weight (M_r) of the LitR protein, 0.1 mg of LitR protein, purified by affinity chromatography, was analyzed using an ÄKTA explorer 10S system equipped with a Superdex 200 10/300 GL column (GE Healthcare). The column was equilibrated with buffer containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, and 200 mM l-arginine monohydrochloride at a flow rate of 0.2 ml/min. l-Arginine monohydrochloride was added to avoid aggregation of LitR (39). Gel filtration calibration kit LMW (GE Healthcare; comprises RNase A at an M_r of 13,700, carbonic anhydrase at an M_r of 29,000, ovalbumin at an M_r of 43,000, and conalbumin at an M_r of 75,000) and gel filtration calibration kit HMW (GE Healthcare; comprises aldolase at an M_r of 158,000 and ferritin at an M_r of 440,000) were used as standard M_r markers. The sample was illuminated with UV-A light (365 nm) at approximately 0.06 μmol · s⁻¹ · m⁻² for 180 s prior to being loaded onto the Superdex 200 10/300 GL column. The relative M_r of LitR was estimated based on the molecular weight markers and results of triplicate individual experiments. Gel filtration chromatography was performed three times using three independent protein preparations. To confirm the eluted protein, proteins were separated by SDS-PAGE and visualized by silver staining.