Histrap column

IPTG induced cell pellet was suspended in 20 mM sodium phosphate buffer pH 7.0 and 500 mM NaCl (buffer A) containing 20 mM imidazole. The cell suspension was homogenised by two cycles of extrusion through French press at 1500 psi. The homogenate was clarified by centrifugation at 15000 r.p.m. for 1 hr and filtered through 0.45 μ membrane. The filtrate was loaded onto 1 mL HisTrap column (Amersham Biosciences) containing precharged high performance nickel sepharose, preequilibrated with buffer A and washed with the same buffer till OD₂₈₀ of the effluent reached the baseline. The bound protein was eluted with a linear gradient of 20–500 mM imidazole contained in the binding buffer. The fractions corresponding to the single major peak were pooled and loaded onto Hi-Load Superdex S200 (Amersham Biosciences) column preequilibrated with 100 mM tris HCl pH 7.5 and 100 mM NaCl. Pure GGT eluted as a single prominent peak and was confirmed by activity test and SDS-PAGE analysis. The protein was concentrated to 10 mg/mL over a 10 kDa cutoff membrane (Pall) and preserved at −20°C.

Escherichia coli strain BL21 (DE3) pET28-PcaY_PP-LBD was grown in 2 l Erlenmeyer flasks containing 500 ml LB supplemented with 50 μg/ml kanamycin at 30°C until an OD₆₆₀ of 0.6, at which point protein expression was induced by adding 0.5 mM isopropyl-beta-D-thiogalactopyranoside. Growth was continued at 18°C overnight and cells were harvested by centrifugation at 10,000 g for 30 min. Cells were then resuspended in buffer A (20 mM Tris/HCl, 200 mM NaCl, 10 mM imidazole, 5 % (v/v) glycerol, 0.1 mM EDTA, pH 8.0) and broken by French Press treatment. After centrifugation at 20,000 g for 15 min, the supernatant was loaded onto a 5 ml HisTrap column (Amersham Bioscience), washed with 10 column volumes of buffer A and eluted with an imidazole gradient of 45–500 mM in the same buffer. Protein containing fractions were pooled and dialyzed overnight at 4°C into analysis buffer (5 mM PIPES, 5 mM MES, 5 mM Tris-HCl, pH 8.0).

E. coli BL21(DE3) containing p2480-LBD was grown in 2-liter Erlenmeyer flasks containing 400 ml LB medium supplemented with 100 μg/ml ampicillin at 30°C. Once the culture reached an optical density at 600 nm (OD₆₀₀) of 0.5, protein overexpression was induced by the addition of 0.5 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG). Growth was then continued at 18°C overnight prior to cell harvest by centrifugation at 6,000 × g for 20 min at 4°C. Cell pellets were resuspended in buffer A (30 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, 10% [vol/vol] glycerol [pH 8.0]) and broken by French press treatment at a gauge pressure of 62.5 lb/in². After centrifugation at 20,000 × g for 1 h, the supernatant was loaded onto a 5-ml HisTrap column (Amersham Biosciences) previously equilibrated with 5 column volumes of buffer A, washed with buffer A containing 40 mM imidazole, and eluted with a linear gradient of 40 to 500 mM imidazole in buffer A. Protein-containing fractions were pooled and dialyzed into HNG buffer (50 mM HEPES, 300 mM NaCl, 10% [vol/vol] glycerol [pH 8.0]) for immediate analysis.

E. coli BL21(AI) containing p2448‐LBD was grown in LB medium supplemented with kanamycin at 28°C. Once the culture reached an OD₆₀₀ of 0.5, protein overexpression was induced by the addition of 0.05 mM isopropyl‐β‐d‐1‐thiogalactopyranoside and l‐arabinose 0.2% (wt/vol). Growth was then continued overnight at 16°C prior to cell harvest by centrifugation at 6000 × g for 30 min at 4°C. Cell pellets were resuspended in buffer A (30 mM Tris.HCl, 300 mM NaCl, 10 mM imidazole, 10% [vol/vol] glycerol, pH 8.0), disrupted by sonication, and centrifuged at 13,000 × g for 1 h. The supernatant was then loaded onto a 5‐ml HisTrap column (Amersham Biosciences) previously equilibrated with 5 column volumes of buffer A, which was subsequently washed with buffer A containing 10 mM imidazole and eluted with a linear gradient of 10 to 500 mM imidazole in buffer A. Protein‐containing fractions were pooled and dialysed into buffer B (50 mM HEPES, 300 mM NaCl, 10% [vol/vol] glycerol, pH 8.0) for immediate analysis.

One milligram of protein dialyzed into the TMP buffer was incubated with one unit of thrombin (Merck) at 16°C for 1.5 h, at which point another unit of thrombin was added and incubated at 16°C for another 1.5 h. The protein solution was loaded onto a 5 mL HisTrap column (Amersham Bioscience) equilibrated with buffer A, washed with five column volumes of TMP buffer and eluted with an imidazole gradient of 45–500 mM in buffer A. Samples were analyzed by SDS‐PAGE. Fractions that correspond to cleaved protein were dialyzed overnight into the TMP buffer for ITC experiments.

E. coli BL21 (DE3) containing the expression plasmids were cultured in LB medium supplemented with 50 μg ml⁻¹ kanamycin at 30 °C until an OD₆₆₀ of 0.6, at which point 0.1 mM IPTG was added. Growth was continued at 18 °C overnight prior to cell harvest by centrifugation at 10,000 × g for 30 min. Pellets were resuspended in buffer A (30 mM Tris, 300 mM NaCl, 10 mM imidazole, 5% (v/v) glycerol, pH 7.0) and broken by French press at 1000 psi. After centrifugation at 20,000 × g for 1 hour, the supernatant was loaded onto a HisTrap column (Amersham Bioscience), washed with buffer A and proteins were eluted with a 45–1000 mM imidazole gradient in buffer A. To eliminate potentially bound compounds, PstS and CtpL-LBD were dialyzed against 10 mM Tris/HCl, 6 M guanidine hydrochloride, pH 8.0. For these experiments highest possible purity reagents were used (note: When the procedure was carried out with PstS and standard purity guanidine hydrochloride, no Pi binding was observed in ITC, which is due to the capture of Pi, present as an impurity in GdnHCl, during protein refolding). Protein was diluted to 10 μM and refolded by two consecutive dialyses into 10 mM Tris/HCl, pH 8.0. Aggregated protein corresponding to misfolded species was removed by filtration using 0.22 μm cut-off filters.

Escherichia coli BL21 (DE3) containing pET28-McpK-LBD was grown in 2 L Erlenmeyer flasks containing 500 ml LB medium supplemented with 50 μg ml^-1 kanamycin at 30°C until an OD₆₆₀ of 0.6, at which point protein production was induced by adding 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG). Growth was continued at 18°C overnight before cell harvest by centrifugation at 10,000 g for 30 min. All subsequent manipulations were carried out at 4°C. Cell pellets were resuspended in buffer A [20 mM Tris/HCl, 200 mM NaCl, 10 mM imidazole, 5% (vol/vol) glycerol, pH 8.0] and broken by French press treatment at 1000 psi. After centrifugation at 20,000 g for 1 h, the supernatant was loaded onto a 5 ml HisTrap column (Amersham Bioscience), washed with five column volumes of buffer A and eluted with a 30–300 mM imidazole gradient in buffer A. Protein-containing fractions were pooled.