A single colony of freshly streaked E.coli BL21 containing pET21d with the desired insert was used to inoculate 5ml LB containing the appropriate antibiotic and incubated overnight at 37⁰C with shaking at 250rpm (15xg). This culture was used to inoculate 100ml (small scale purification) or 1L (large scale purification) to an
absorbance of 0.05 at a reading of OD600 and was incubated at 37⁰C with shaking until
the absorbance reached 0.4-0.6. 1mM IPTG was then added and the culture incubated for a further 3hr. Cells were harvested by centrifugation at 5100rpm (5525xg) for 10min at 4⁰C and the supernatant discarded. The resulting pellet was stored at -20⁰C.
2.14.2 Analysis of recombinant protein stability
During the preparation of cells in section 2.17.1, 1ml of culture was removed before the addition of IPTG (uninduced sample), and 2 samples of 1ml of culture was removed following the 3hr incubation after the addition of IPTG (induced sample). The cultures were centrifuged at 13000rpm (10000xg) for 5min at room temperature to harvest the cells, and the supernatant was discarded. The uninduced sample was resuspended in 100μl SDS loading buffer and one of the induced samples was resuspended in 250μl SDS loading buffer. The samples were boiled for 5min and then centrifuged at
13000rpm (10000xg) for 3min to sediment any insoluble material. 15μl of each sample were analysed by SDS-PAGE to confirm overexpression of the recombinant protein. To determine the solubility of the recombinant protein, the second induced sample was resuspended in 250μl START buffer and lysozyme was added to a final
concentration of 1mg ml-1. The sample was incubated at room temperature for 60min and then sonicated for 3 x 10s using a Sanyo soniprep 150. The sample was then centrifuged at 13000rpm (10000xg) for 10min allowing the separatation of soluble and insoluble material. The supernatant, containing soluble proteins, was transfered into a
66 fresh eppendorf and 250μl SDS loading buffer was added. The pellet, containing
insoluble material, was resuspended in 250μl START buffer 8M urea and 250μl SDS loading buffer was added. Both samples were boiled for 5min, centrifuged at 13000rpm (10000xg) for 3min and 15μl of each sample was analysed by SDS-PAGE.
2.14.3 Separation of insoluble and soluble proteins.
Cells from section 2.17.1 were resuspended in 5ml START buffer without urea. The suspension was then freeze-thawed three times by placing the sample at -80⁰C for 10min and then allowing it to thaw completely on ice. The cells were then broken by sonicating (using a Sanyo soniprep 150) ten times for 10s with 1min rest on ice in between each run. The suspension was centrifuged at 10000rpm (25000xg) for 25min at 4⁰C. The supernatant containing soluble proteins was removed, filtered using a 0.45μm pore size, and stored at 4⁰C. If the recombinant protein had been determined to be insoluble (Section 2.12.2), the pellet was resuspended thoroughly in 5ml (small scale purification) or 30 ml (large scale purification) START buffer 8M urea and incubated overnight at 4⁰C. The sample was then centrifuged (10000rpm (25000xg), 25min, 4oC) to remove any unbroken cells and the resulting supernatant containing the solubilised proteins was filtered using a 0.45μm pore size and stored at 4⁰C.
2.14.4 HiTrap purification
A 5ml HiTrap column (GE Healthcare) was prepared by washing with 10ml sdH2O, and
then charged with 10ml 50 mM NiSO4. The column was washed with 10ml sdH2O to
remove excess NiSO4. The BioRad Econo Gradient pump and Fraction Collector was
flushed with START buffer at a flow rate of 1.5ml min-1. When purifying insoluble proteins as determined in Section 2.17.3, 8M urea was added to both the START and elution buffer. The charged HiTrap column was attached to the BioRad Econo Gradient pump and equilibrated by washing with 5ml START buffer. The supernatant containing recombinant protein was applied to the column at a flow rate of 1ml min-1 and any non-specific proteins were removed by washing with 5% elution buffer until the absorbance returned to zero. The his-tagged proteins were eluted from the column using a gradient of 5-100% elution buffer (containing 0.5 M imidazole) at a flow rate of 1ml min-1, with 1ml fractions being collected. Imidazole has a higher affinity for the
67 nickel than the His-tag displacing recombinant protein. The eluted fractions were then analysed by SDS-PAGE. After elution of the recombinant protein, the HiTrap column was washed with 10ml 50mM EDTA to remove NiSO4, followed by 10ml sterile water.
10ml 20% (v/v) ethanol was washed through the column before storage at 4oC.
2.14.5 Protein dialysis
2.14.5.1 Preparation of dialysis membrane
Size 2 dialysis membrane tubing (Medicell International) was used, which allows selection of proteins larger than 12kDa. Before use, the dialysis tubing was boiled in 2mM EDTA for 20min and washed thoroughly in sdH2O. For long term storage, the
EDTA-boiled dialysis tubing was placed in 50% (v/v) ethanol and stored at 4oC.
2.14.5.2 Dialysis of recombinant protein
Fractions containing recombinant protein (Section 2.12.4) were placed in dialysis membrane tubing and dialysed in either START buffer, 20mM Tris-HCl pH7.5 plus 0.15M NaCl for 12hr at 4oC. This dialysis step was repeated twice more, replacing the dialysis solution with fresh appropriate buffer each time. If the protein was insoluble, 4 M urea was added to the dialysis solution during the first dialysis. The solution was then replaced by the appropriate buffer containing 2M urea and dialysis carried out for 12hr at 4oC. This was repeated three times using the appropriate buffer containing 1M, 0.5M and 0M urea. After dialysis, the protein fractions were removed from the dialysis tubing and glycerol was added to a final concentration of 10% (v/v). 200μl aliquots of recombinant protein were stored at -70oC.