Affinity column

The soluble lysate proteins were analysed on 12% SDS gel for confirmation of recombinant proteins and protein lysis. Lysate was purified using affinity column (Bio-RAD, CA, USA) and 6X histidine-tagged protein was bound to NI-NTA resin (Invitrogen, USA) and incubated at 12 °C for 2 h. The column was washed with 10 column volumes of wash buffer containing 40 mM imidazole and was eluted with a buffer containing 200 mM imidazole. Similarly, GST-tagged protein was bound to glutathione sepharose resin (GST, Takara, Japan) and eluted with 20 mM reduced glutathione. The eluted fractions were then dialysed and analysed by SDS-PAGE and WB.

The Cb coding sequence with a C-terminal 6-His tag was cloned into a pET22b bacterial expression vector and transfected into chemically competent Rosetta (DE3) bacteria (EMD Millipore). A 100 ml culture of LB-ampicillin was grown for 24 hr from a single bacterial colony. 25 mls of bacteria was used to inoculate 500 mls of LB-ampicillin and grown at 37°C until OD between 0.6 and 0.8. Expression was induced with 0.5 µM IPTG at 37°C for 4 hr. Bacterial pellets were frozen overnight at −80°C. Bacteria pellets were resuspended in 40 mls of xTractor buffer (Clontech) and then sonicated for 4 min on ice. Extracts were clarified at 9500x g for 20 min at 4°C. Supernatant were added to 1 ml of equilibrated Talon resin and agitated at 4°C for 60 min. Supernatant/resin mix was added to affinity column (Biorad) and supernatant allowed to flow through by gravity. Resin was washed with 20mls of Talon equilibration buffer (Clontech) followed 10 mls wash buffer (Equilibration buffer with 1/10th volume of elution buffer). Proteins were eluted with 10mls of elution buffer in 1 ml aliquots. Elutions containing rCb were pooled and dialyzed overnight in 125KMEI buffer (125 mM KCl, 1 mM MgCl₂, 1 mM EGTA, 10 mM Imidazole pH 7.0, 10 mM DTT). All steps were performed at 4°C and proteins were stored at 4°C. Protein concentration was determined using Bradford Assay.