Purification of His-PBP2a variants

E. coli Rosetta2 (DE3 pLysS) was transformed with pCCM97 [His-pbp2a(A77T)], pCCM98 [His-pbp2a(S410A)], pCCM99 [His-pbp2a(E131A)], or pMFS8 [His-pbp2a(WT)]. Fresh transformants were precultured in terrific broth (TB) supplemented with Kan (25 µg/mL) and Cm (25 µg/mL). Cultures were grown at 37°C to an OD₆₀₀ of 0.4 and then used to inoculate 1 L of TB supplemented with antibiotics at an OD₆₀₀ of 0.01. The cultures were grown at 37°C to an OD₆₀₀ of 0.4, at which time they were cooled to room temperature (RT). IPTG (isopropyl thiogalactopyranoside) was added to 1.5 mM final concentration, and the cultures were grown overnight at 18°C. Cells were harvested by centrifugation at 4,667 × g for 15 min, and pellets were weighed and frozen. Cell pellets were resuspended in 10× the pellet volume with buffer A (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% glycerol, 2× complete protease inhibitors), sonicated, and further lysed by three passes through a cell disruptor at 25,000 PSI at 4°C (Constant Systems). The lysates were subjected to ultracentrifugation at 200,000 × g for 1 hour, at 4°C. The membrane pellets were dounce homogenized in 30 mL of buffer B (20 mM Tris-HCl at pH 7.4, 150 mM NaCl, 10% glycerol, 1% wt/vol CHAPS). The homogenate was rolled for 1 hour at 4°C, followed by ultracentrifugation at 200,000 × g for 1 hour, at 4°C. The detergent-solubilized membrane proteins (S100) were passed through a 0.22 PES filter prior to purification on the AKTA purifier system using a HisTrap column (1 mL, GE). After loading and washing with buffer C (20 mM Tris-HCl at pH 7.4, 500 mM NaCl, 10% glycerol, 0.1% reduced Triton-X-100), the sample was eluted with a gradient of buffer D (20 mM Tris-HCl at pH 7.4, 500 mM NaCl, 10% glycerol, 0.1% reduced Triton-X-100, 500 mM imidazole). Peak fractions were pooled and dialyzed overnight at 4°C in buffer C. The proteins were analyzed for purity by SDS-PAGE using 4%–20% polyacrylamide gels stained with InstantBlue (Expedeon).