Recombinant Protein Expression and Purification

Recombinant protein expression and purification was carried out as previously described (10 (link)). Briefly, E. coli BL21 (DE3) transformed with the plasmid for GLuc or EC1-GLuc was grown in a LB medium containing 100 μg/ml ampicillin at 37°C. When the OD₆₀₀ reached 0.6, the expression of the recombinant proteins was induced by the addition of 0.2 mM isopropyl 1-thio-β-D-galactopyranoside at 25°C overnight. The GLuc-6His and EC1-GLuc-6His proteins were purified from the supernatant using ProBond Nickel-chelating resin (Invitrogen). The recombinant proteins were subjected to 15% SDS-PAGE, and the expression was confirmed by Coomassie brilliant blue staining and western blot analysis with rabbit anti-GLuc serum (1:1,000; Nanolight Technology, Pinetop, AZ, USA). The proteins were finally dialyzed against PBS (pH 7.4) at 4°C for 24 h, and the concentrations were determined using a Biadford protein assay kit (Bio-Rad). Aliquots of protein were stored at −80°C prior to use.