The E. coli BtuCD Structure: A Framework for ABC Transporter Architecture and Mechanism Kaspar P. Locher, Allen T. Lee, and Douglas C. Rees

The sequences for the different transporters were subcloned into various pET vectors (Novagen), which attached poly-histidine tags at the amino- or carboxy-terminus. The constructs were expressed in E. coli BL21 (DE3) (Stratagene) in a 50-liter fermenter at 37°C and were induced using 1 mM IPTG. BtuC and BtuD were coexpressed from a single plasmid, with the BtuC subunit containing an NH₂-terminal decahistidine tag. BtuCD protein was solubilized in 1% dodecyl-N,N-dimethylamineoxide (LDAO) and purified using Ni-NTA metal affinity chromatography (Qiagen), followed by gel filtration. Crystals were grown by mixing BtuCD protein (20 mg/ml) with an equal volume of reservoir solution (100 mM Tris pH 8.0, 300 mM magnesium nitrate, 21% polyethylene glycol 2000, 0.8% 2-methyl-2,4-pentanediol in D₂O) in sitting drops. Crystals grew to a final size of 0.1 0.15 0.5 mm³ in a week, and were frozen in liquid N₂ before data collection. Cells expressing selenomethionine BtuCD protein were grown in M9 medium supplemented with seleno-D,L-methionine. Purification and crystallization of the selenomethionine protein was as described above for the native protein. Data sets were collected at the Advanced Photon Source (APS) or at the Stanford Synchrotron Radiation Laboratory (SSRL).