Superdex 75 10 300 gl

Absolute molar mass was calculated using the ÄKTAmicro system (GE Healthcare) coupled with a Dawn HELEOS II MALLS detector and an OptiLab T-rEX online refractive index detector (Wyatt Technology). Five hundred microliters of protein sample was injected onto the Superdex 200 10/300 GL HPLC size exclusion column (Cytiva) for DfrB-H5 and Superdex 75 10/300 GL (Cytiva) for DfrB1 at a flow rate of 0.4 ml min⁻¹. Bovine serum albumin was used for calibration.

All steps were carried out at 4 °C. Frozen cells were thawed and lysed by sonication in buffer A supplemented with 0.1 mM phenylmethanesulfonyl fluoride. The lysate was then supplemented with 0.5 U/mL benzonase nuclease and centrifuged at 18,000 rpm for 40 min. The supernatant was filtered with a 0.45-μm syringe filter, then loaded onto a 5-mL Ni-smart beads 6FF FPLC column (Smart-Lifesciences, Changzhou), and eluted with a linear gradient of imidazole using buffer A and buffer B (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 M Imidazole). Peak fractions were collected and pooled for dialysis in buffer C (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM DTT) overnight to remove imidazole. Dialyzed sample was centrifuged to remove any possible precipitates, and the supernatant was loaded onto a 5-mL Heparin beads 6FF FPLC column (Smart-Lifesciences, Changzhou) pre-equilibrated with buffer C, and then eluted with a linear gradient of buffer C and buffer D (20 mM Tris-HCl, pH 7.5, 1 M NaCl, 1 mM EDTA, 1 mM DTT). Peak fractions were pooled and concentrated, and then loaded onto a Superdex 75 10/300 GL (Cytiva) column pre-equilibrated in buffer C. Peak fractions were collected and analyzed by SDS-PAGE. The AAG proteins were concentrated to 1 mg/mL and stored at –80 °C in small aliquots.

HMW LPS was separated from LMW LPS by size exclusion chromatography using a Superdex 75 10/300GL (Cytiva) under dissociative conditions in the presence of 0.25% sodium deoxycholate, pH 9.2 (62 (link)). The eluting fractions were monitored with refractive index (Shimadzu RID-10A) and with DOC-PAGE stained with alcian blue and silver (see Text S1 for further details on the DOC-PAGE method). The O-PS (polysaccharide portion of LPS, including core, O-antigen, and galactan) was released from the lipid A by using 1% acetic acid for 1.5 to 2 h at 100°C until the formation of insoluble lipid A precipitate. The lipid A precipitate was removed from the O-PS soluble fraction by initial centrifugation for 25 min at 3,500 × g. The pellet was washed by addition of water, resuspension, and centrifugation at 100,000 × g for 4 h at 4°C. The soluble O-PS fraction was also ultracentrifuged at 100,000 × g for 4 h, 4°C, and the supernatant was freeze-dried. The dry O-PS was dissolved in water and passed through a 0.22-μm nylon filter to remove any trace of free lipids, LPS, or lipid A and was used for structural work.