The avian reovirus S1133 protein σA was expressed as a maltose-binding-protein fusion using the expression vector pMALCS2 described in Gonzalez-Lopez et al.
). Prior to proceeding with milligram-scale expression and purification for crystallization experiments, the sequence of the insert encoding σA was confirmed by DNA-sequence analysis (Sistemas Genomicos, Valencia, Spain).
For large-scale expression, the Escherichia coli strain XL1-Blue was freshly transformed with the pMALCS2 vector and four 750 ml cultures were grown aerobically at 310 K to an optical density of 0.6 measured at 600 nm in LB medium. Expression was induced by adding 1 mM isopropyl β-d-thiogalactopyranoside and allowed to proceed for 3 h. Harvested cells were resuspended in 40 ml lysis buffer (20 mM Tris–HCl pH 7.3, 200 mM sodium chloride, 1 mM dithiothreitol) containing a protease-inhibitor cocktail specific for bacterial cell extracts (Sigma–Aldrich, Madrid, Spain) and the suspension was frozen at 253 K. The cellular pellet was lysed by a double pass through an emulsifier (Avestin Emulsifier C5, Avestin Europe GmbH, Mannheim, Germany). After removal of the insoluble material by centrifugation, sodium chloride and poly(ethyleneimine) were added to final concentrations of 1 M and 0.1%(w/v), respectively, and the suspension was incubated at 277 K for 1 h to precipitate nucleic acids. Following a second centrifugation step, bovine ribonuclease A (>70 Kunitz units per milligram; Sigma–Aldrich, Madrid, Spain) was added to the supernatant to a final concentration of around 30 µg ml−1 and incubated for 30 min at room temperature. 3 ml suspended amylose resin (New England Biolabs, Ipswich MA, USA) was then added and gently mixed and the suspension was left for 10 min at room temperature before being poured into an empty column. The resin was washed three times with three column volumes of MBP buffer (20 mM Tris–HCl pH 7.5, 200 mM sodium chloride, 1 mM EDTA) and elution was performed by washing twice with 10 ml 10 mM maltose in MPB buffer. Calcium chloride and protease factor Xa (New England Biolabs, Ipswich MA, USA) were added to final concentrations of 1 µg ml−1 and 1 mM, respectively. Incubation was carried out at room temperature for 20 h. The digested protein was dialysed overnight against TE buffer (10 mM Tris–HCl pH 8.5, 1 mM EDTA) and applied onto a 1 ml Uno-Q column (Biorad, Barcelona, Spain). Pure σA eluted in the flowthrough, while maltose-binding protein and other impurities were retained on the column. The protein was concentrated with Centricon concentrators (Millipore, Madrid, Spain), using three washes with TE buffer to eliminate small-molecule impurities.
Crystallization was carried out by sitting-drop vapour diffusion at 278 K in CompactClover plates (Jena Biosciences, Jena, Germany), with 0.1–0.15 ml reservoirs and drops consisting of 2–5 µl protein solution (14–30 mg ml−1
protein in TE buffer) mixed with 2–5 µl reservoir solution, using equal volumes of both. Diffraction images were processed using MOSFLM
) and the crystallographic data were scaled using SCALA
(Collaborative Computational Project, Number 4, 1994
). Self-rotation functions were calculated using MOLREP
(Vagin & Teplyakov, 2000