Pfs25 Binding and Characterization with Hydroxocobalamin

His-tagged Pfs25, prepared from a Baculovirus system as previously described [31 (link)], was diluted to 80 µg/mL and incubated with varying amounts of hydroxocobalamin hydrochloride. At the optimum binding ratio, hydroxocobalamin hydrochloride (320 µg/mL) was incubated with 80 µg/mL Pfs25 at different time points and then subjected to native electrophoresis using Tris-Glycine gel (Lonza, Basel, Switzerland). Hydroxocobalamin acetate (Sigma-Aldrich # H8017) and cyanocobalamin were also incubated with Pfs25 for comparison at 1:4 Pfs25:Cbl/CoPoP binding ratio. For peptide binding, a fluorescently labeled peptide, FAM-RGD, was used and was previously demonstrated to bind to CoPoP liposomes [14 (link)].