Construct and protein production

Construct for protein crystallisation:

Protein expression and purification (done by M. Fairhead @mfj399):

Expression and purification were performed using the PREPX workflow, described below. Typically, 100 ng of plasmid was transformed into the E. coli protein expression strain BL21(DE3), and a single colony was used to inoculate 10 mL of SOC containing 50 µg/mL kanamycin. The starter culture was grown overnight at 37°C 250 rpm shaking, and was used the following day to inoculate 1 L of TB (ForMedium) in a 2.5 L baffled shake flask supplemented with 50 µg/mL kanamycin and 0.01% Antifoam-204 (Merck). Cells were grown first for 3 h at 37°C and then for 2 h at 18°C, 250 rpm shaking. Protein expression was then induced with 0.5 mM Isopropyl-β-D-thiogalactopyranosid (IPTG), and growth continued overnight at 18°C, 250 rpm shaking.

Cells were harvested by centrifugation at 4,000xg and the pellets were re-suspended using 3 mL of Base Buffer (10 mM HEPES, 500 mM NaCl, 5 % glycerol, 30 mM imidazole, 0.5 mM TCEP, pH 7.5) per gram wet weight of cells. Triton X-100, lysozyme and benzonase to a final concentration of 1 %, 0.5 mg/mL and 1 µg/mL, respectively, were then added before freezing at -80°C. The cell lysate mixture was first thawed in a room-temperature water bath and then centrifuged at 4800xg for 1 h at 4°C. The supernatant (soluble fraction) was then loaded onto a 1 mL His GraviTrap column (Cytiva) and washed twice with 20 mL of Base Buffer. Four His GraviTrap columns were used for every 1 L of culture originally grown. The tagged-target protein was then eluted using 2.5 mL of Base Buffer containing 500 mM imidazole and immediately desalted using a PD-10 column (Cytiva) with Base Buffer as the mobile phase. The His tag was then removed by protease digest overnight at 4°C using 1 mg of TEV per 10 mg of target protein. The tag, un-cleaved protein and TEV (bearing a non-cleavable his-tag) were then removed by passing through a 1 mL His GraviTrap column. The cleaved protein sample was then injected onto a superose 12 pg SEC column (Cytiva) using 25 mM Tris, 100 mM NaCl, 5 % glycerol, pH 7.5, as the mobile phase. Peak nsP3 macrodomain fractions were concentrated to 11 mg/mL before flash freezing in liquid nitrogen and storage at -80°C. Efficacy of the process and final protein purity were assessed by SDS-PAGE using NuPAGE 4-12 % Bis-Tris Midi Protein Gels (ThermoFisher Scientific).