PLACA INFERIOR DE PRENSADO

2.2.11.1 Production and Purification o f GST-fusion Proteins

For a 500ml prep, 50ml LB + ampicillin was inoculated with a fresh bacterial colony containing the plasmid o f interest and grown overnight at 37°C. This culture was diluted 1:10 with 450ml LB + ampicillin and incubated for 1 hour at 37°C. IPTG was added to a final concentration o f O.lmM and the culture grown for a further 2-4 hours at 37°C, or 5-7 hours at 30°C, to induce expression o f the recombinant protein. The cells were then chilled on ice and PMSF added to a final concentration o f O.lmM. After pelleting the cells by centrifugation at 5,000g- for 5 minutes at 4°C, the supernatant was discarded and the cell pellet was frozen at -70°C, then thawed quickly

at 37°C. Cells were resuspended in 10-20ml ice-cold NETN buffer and lysozyme was added to 2mg/ml final (except for cells carrying the pLysS gene). After a 10 minute incubation on ice with occasional shaking, the sample was sonicated on ice 3 times for 15 seconds each to complete cell lysis and to shear DNA. The lysate was centrifuged at 12,000g for 15 minutes at 4°C to pellet cell debris and the supernatant transferred to a fresh tube. After a further 15 minutes spin, KCl was added to 200mM final (to improve binding to the beads) and 400pl glutathione-agarose or glutathione-sepharose beads (pre-blocked in 1% BSA for 15 minutes, room temperature, then washed 4 times in NETN and stored as a 50% slurry) were added. The beads and lysate were rotated for 1-2 hours at 4°C then briefly spun in a centrifuge to pellet the beads. These were then washed 4 times with NETN and stored at -80°C as a 50% slurry in PSB. A lOpl aliquot was removed for SDS-PAGE analysis.

For insoluble GST fusion proteins, sarkosyl was added to 1.5% after sonication to solubilise the protein. Before binding to beads, 2% NP-40 was added to sequester the sarkosyl (Frangioni and Neel, 1993).

NETN: 20mM Tris-Cl (pH8.0), lOOmM NaCl, Im M EDTA,

0.5% NP-40, ImM DTT, protease inhibitors

PSB: lOmM Hepes (pH 7.6), lOOmM NaCl, Im M EDTA, 25%

Glutathione Elution

GST fusion proteins were eluted from beads by competing with glutathione. Beads were washed in Elution buffer, then resuspended in an equal volume o f Elution buffer + 20mM glutathione (freshly prepared). After a 10 minute incubation on ice with occasional agitation, beads were spun down and the supernatant, containing eluted protein, was collected. This elution step was repeated twice more, and the supernatants pooled. To remove contaminating beads, the supernatant was spun at 5,000g for 1 minute, then carefully transferred to a Microcon-10 column (Amicon) and spun through a particle separator filter (Amicon).

Elution buffer: 50mM Tris-Cl (pH 8.0), lOOmM NaCl, 5mM DTT (fresh), 0.1% NP-40, protease inhibitors

Thrombin Digestion

Proteins were cleaved from the GST by digestion with thrombin (in constructs with thrombin cleavage sites). Beads were washed 3 times in PBS, then once in PBS + 2.5mM CaCl]. After resuspending in 4 volumes o f PBS + CaCE, thrombin was added (0.2-1% w/w). Beads were rotated for 1-4 hours at room temperature, then spun at 5,000g for 1 minute to pellet the beads. The supernatant, containing the cleaved, released protein, was collected. The beads were washed with 1 volume o f PBS, this was collected as before and pooled with the previous supernatant. 50pg/ml BSA and

10% glycerol were added when storing the protein at -80°C.

2.2.11.2 Production and Purification o f His-tagged Proteins

For a 500ml prep, 50ml LB + kanamycin was inoculated with a fresh bacterial colony containing the plasmid o f interest and grown overnight at 37°C. This culture

was diluted 1:10 with 450ml LB + kanamycin and incubated for 1 hour at 37°C. IPTG was added to a final concentration o f Im M and the culture grown for a further 2-4 hours at 37°C, or 5-7 hours at 30°C, to induce expression o f the recombinant protein. The cells were then chilled on ice and PMSF added to a final concentration o f O.lmM. After pelleting the cells by centrifugation at 5,000g for 5 minutes at 4°C, the supernatant was

were resuspended in 10-20ml ice-cold His-Wash buffer and lysozyme was added to 2mg/ml final (except for cells carrying the pLysS gene where this step was unnecessary). After a 10 minute incubation on ice with occasional shaking, the sample was sonicated on ice 3 times for 15 seconds each to complete cell lysis and to shear DNA. The lysate was centrifuged at 12,000g for 15 minutes at 4°C to pellet cell debris and the supernatant transferred to a fresh tube. After a ftirther 15 minutes spin, lOOpl Ni-NTA-agarose beads (pre-blocked in 1% BSA for 15 minutes, room temperature, then washed 4 times in His-Wash buffer and stored as a 50% slurry) were added. The beads and lysate were rotated for 1-2 hours at 4°C then briefly spun in a centriftige to pellet the beads. These were then washed 4 times with His-W ash buffer and stored at - 80°C. A lOpl aliquot was removed for SDS-PAGE analysis.

His-Wash buffer: 20mM Tris-Cl (pH 8.0), 400mM NaCl, lOmM imidazole, 0.5% Tween-20, protease inhibitors (no EDTA or DTT)

Elution o f His-tagged proteins

To elute His-tagged proteins from Ni-NTA-agarose beads, an equal volume o f Elution buffer containing 200mM imidazole (fresh) was added to the beads and incubated on a rotating wheel at 4°C for 5 minutes. Beads were collected by a brief spin at l,000g for 30 seconds and the supernatant containing eluted protein was collected and kept on ice. Further elution steps were then performed on the same beads as before, using increasing amounts o f imidazole: 400mM, 600mM and IM . Aliquots o f the eluted protein were analysed by SDS-PAGE. Eluted protein was snap-frozen in liquid nitrogen and stored at -80°C.

Elution Buffer: 50mM Tris-Cl (pH 7.5), 250mM NaCl, 20% glycerol, 0.2-IM imidazole, protease inhibitors (no EDTA or DTT)