Superdex 200 10 300 gel filtration column

The absolute molar mass of bsIRG1 in solution was determined by MALS. The target protein filtered with a 0.2 µm syringe-filter was loaded onto a Superdex 200 10/300 gel-filtration column (GE Healthcare) that had been pre-equilibrated in buffer comprising 20 mM Tris–HCl (pH 8.0) and 150 mM NaCl. The mobile phase buffer flowed at a rate of 0.4 mL/min at room temperature. A DAWN-treos MALS detector (Wyatt Technology, Santa Barbara, USA) was connected with the ÄKTA explorer system (GE Healthcare). The molecular mass of bovine serum albumin was used as a reference value. Data for the absolute molecular mass were assessed using the ASTRA program (Wyatt Technology).

Small angle X-ray scattering data were collected at the Australian Synchrotron on the SAXS/WAXS beamline. The X-ray beam size at the sample was 250 μm horizontal, 150 μm vertical and data were collected using a Pilatus 1 M detector positioned 1600 mm from the sample, giving a q range of 0.006–0.4 Å⁻¹. Protein samples alone were subjected to in-line size exclusion chromatography on a Superdex 200 5/150 GL gel-filtration column (GE Healthcare) with a bed volume of 3 ml. Protein complexes were separated on a Superdex 200 10/300 gel-filtration column (GE Healthcare) with a bed volume of 24 ml. 50 μl sample or 100 μl complexes at 8–10 mg/ml were injected and the fractionated sample flowed through a 1.5 mm quartz capillary with a co-flow system^{64 (link)} where it was exposed to the X-ray beam. Data were collected at 16 °C with 500 detector images of sequential 1 and 5 s exposures for the 5/150 and 100/300 columns respectively.
Radial averaging, background subtraction and image series analysis was performed using scatterBrain (software package developed at the Australian Synchrotron). Five sequential images were averaged to generate each SAXS data set before subsequent analysis using the ATSAS 2.5.0 software^{65 (link)}. Guinier fits and Kratky plots were made using PRIMUS and P(r) distribution analysis was performed using GNOM.

The gene encoding the full length msmeg_1954 was amplified by polymerase chain reaction (PCR) from M. smegmatis genomic DNA and sub-cloned into prokaryotic expression vector pGEX-6p-1 with an N-terminal GST tag. Recombinant plasmid was transformed into E. coli BL21-Codon Plus (DE3) RIL for protein expression.
Cells were grown in Luria Bertani medium supplemented with 100 mg/L ampicillin at 37°C until OD₆₀₀ reached 0.8 and then induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 18 h at 16°C. Cells were harvested by centrifugation and lysed in 1 x PBS, pH 7.4 by ultrasonication. After centrifugation of cell lysates, supernatants containing soluble recombinant protein were loaded onto a GST-affinity chromatography (GE Healthcare) and then washed with 1 × PBS to remove non-specifically bound proteins. The proteolytic cleavage of the GST-tag was performed with PreScission Protease overnight in cold room, and the tagless MSMEG_1954 was further purified with a Superdex 200 (10/300) gel filtration column (GE Healthcare) pre-equilibrated with a buffer containing 25 mM Tris-HCl, pH 8.0, and 150 mM NaCl. Fractions containing target protein were then pooled and concentrated for crystallization. The expression and purification of MSMEG_1954 mutants used in the ATPase assay were carried out similarly as described above.

CB1 was expressed and purified as described previously^{13 (link)}. Briefly, human full-length CB1 containing an N-terminal FLAG tag and C-terminal histidine tag was expressed in Spodoptera frugiperda Sf9 insect cells with the baculovirus method (Expression Systems). Receptor was extracted using 1% lauryl maltose neopentyl glycol (L-MNG) and purified by nickel-chelating Sepharose chromatography. The eluant from the Ni column was applied to an M1 anti-FLAG immunoaffinity resin. After washing to progressively decreasing the concentration of L-MNG, the receptor was eluted in a buffer consisting of 20 mM HEPES pH 7.5, 150 mM NaCl, 0.05% L-MNG, 0.005% cholesterol hemisuccinate (CHS), FLAG peptide and 5 mM EDTA. Finally, CB1 was purified with size exclusion chromatography, on Superdex 200 10/300 gel filtration column (GE) in 20 mM HEPES pH 7.5, 150 mM NaCl, 0.02% L-MNG, 0.002% CHS. Ligand-free CB1 was concentrated to ~500 µM and stored at −80 °C.

For production of C-terminally His₈-tagged Glt_Tk, E. coli MC1061 containing a pBAD24 derived plasmid were grown in LB medium with 100 µg/mL ampicillin at 37 °C, 200 r.p.m. When the OD₆₀₀ reached 0.8 expression was induced with 0.05% l-arabinose for 3 h. Cells were harvested by centrifugation (15 min, 7400 × g, 4 °C) and resuspended in 20 mM Tris-HCl, pH 8.0. After breaking cells (25 kPsi, 5 °C, Constant Systems Ltd. Daventry UK) the membrane fraction was collected by ultracentrifugation of the supernatant (90 min, 193,360 × g, 4 °C), resuspended in 20 mM Tris-HCl, pH 8.0, and stored at −80 °C. To obtain apo-Glt_Tk Na⁺ was omitted from all buffers. An aliquot of membrane vesicles representing ~1.2 g cells was solubilized in 50 mM Tris-HCl, pH 8.0, 300 mM KCl, 1% n-dodecyl-β-d-maltoside (DDM) for 1 h at 4 °C. After ultracentrifugation (30 min, 265,000 × g, 4 °C) the supernatant was incubated with Ni-Sepharose resin (GE Healthcare) for 1 h at 4 °C. The column was washed with 50 mM Tris HCl, pH 8.0, 300 mM KCl, 0.15% n-decyl-β-d-maltoside (DM), 60 mM Imidazole, pH 8.0, and the protein was eluted with the same buffer containing 500 mM Imidazole. Glt_Tk was further purified by size exclusion chromatography on a Superdex 200 10/300 gel-filtration column (GE Healthcare) in 10 mM Hepes KOH, pH 8.0, 100 mM KCl, 0.15% DM.