Recombinant PfRH5, sourced from Prof. Simon J Draper (University of Oxford, Oxford, UK), was expressed using a Drosophila melanogaster Schneider 2 stable cell line system as previously described [49 (link)].
Recombinant extracellular domains of merozoite antigens PfMSRP5-bio-his (Addgene plasmid #50805), PfSERA9-bio-his (Addgene plasmid #50820), PfRAMA-bio-his (Addgene plasmid #50737), and PfCyRPA-bio-his (PFD1130w-bio-his, Addgene plasmid #50823) were gifts from Dr. Gavin Wright (University of Oxford, Oxford, UK) [45 (link)]. Transient transfection of expression plasmids was performed using the Expi293FTM Expression System Kit (ThermoFisher) as per manufacturer’s instructions. Media was harvested four days post transfection for purification. This supernatant was then passed through a 5 mL HisTrap HP chromatography column (GE Healthcare) charged with 0.1M NiSO4.6H2O and washed with sodium phosphate buffer with 25 mM Imidazole. Proteins were eluted from the column by passing through 500 mM imidazole in sodium phosphate buffer. Elution fractions were pooled and buffer exchanged into PBS. Quality of antigens were assessed using SDS-PAGE and western blotting performed with Anti-his-HRP (C-term) antibody (Miltenyi Biotec).
Recombinant extracellular domains of merozoite antigens PfMSRP5-bio-his (Addgene plasmid #50805), PfSERA9-bio-his (Addgene plasmid #50820), PfRAMA-bio-his (Addgene plasmid #50737), and PfCyRPA-bio-his (PFD1130w-bio-his, Addgene plasmid #50823) were gifts from Dr. Gavin Wright (University of Oxford, Oxford, UK) [45 (link)]. Transient transfection of expression plasmids was performed using the Expi293FTM Expression System Kit (ThermoFisher) as per manufacturer’s instructions. Media was harvested four days post transfection for purification. This supernatant was then passed through a 5 mL HisTrap HP chromatography column (GE Healthcare) charged with 0.1M NiSO4.6H2O and washed with sodium phosphate buffer with 25 mM Imidazole. Proteins were eluted from the column by passing through 500 mM imidazole in sodium phosphate buffer. Elution fractions were pooled and buffer exchanged into PBS. Quality of antigens were assessed using SDS-PAGE and western blotting performed with Anti-his-HRP (C-term) antibody (Miltenyi Biotec).
Free full text: Click here
