| Pelleted cells were resuspended in 20mM TrisHCl (pH 7.9), 150mM NaCl and lysed by sonication. Inclusion bodies were pelleted by centrifugation at 12000g and solubilized in solubilization buffer. Following centrifugation, the supernatant was loaded onto a nickel affinity column, which was then washed with 10 column volumes of wash buffer containing 20mM imidazole. The protein was then eluted in wash buffer containing 1M imidazole. The eluted fractions were then pooled and dialized firstly in H2O and then 0.5% acetic acid to remove imidazole and nickel ions.
Following dialysis, the precipiated proteins were solubilized in 8M urea and reduced with 50mM dithioerythritol (DTE) (pH 4.0) at 37degC for 30min. The protein solution was neutralized, and the pH adjusted to 10.0 with 50mM sodium carbonate/bicarbonate with 1mM EDTA. The protein was then concentrated to 0.5mg/ml and dialyzed gainst 100 volumes of refolding buffer. After 8h, the buffer was changed once and 1mM cystamine was added to facilitate disulfide bond formation.
The refolded protein mixture was adjusted to pH 7.0, then loaded onto a 5mL HiTrap Blue Sepharose column preequilibrated witn 50mM potassium phosphate buffer (pH 7.0). After washing with 10 column volumes of buffer, the protein was eluted wtih 50mM potassium phosphate buffer (pH 7.0) containing 1.5M NaCl. The collected fractions were concentrated to 10mg/ml by ultrafiltration. At this stage, the His-tag may be removed by thrombin cleavage if required. The protein was then further purified on a Superdex75 column with 50mM sodium phosphate (pH 6.5), 150mM NaCl, 0.1mM EDTA and 0.02% sodium azide.
To remove lipopolysaccharides or hydrophobic ligands copurified with the protein a delipidation step was performed. The protein solution was adjusted to pH 3.0 with H3PO4. Then hydroxyalkoxypropyl dextran type VI resin (Sigma) was added to 10mg/ml protein aliquots at a ration of 10:1(w/w) dextran:protein. The dextran and protein suspensions were mixed thoroughly with rotation at room temperature for 90min. The protein was then separated from the resin by centrifugation, and dialyzed against 50mM sodium phosphate (pH 6.5), 150mM NaCl, 0.02% sodium azide, 0.1mM EDTA. |