Anti his hrp c term antibody

The expression vector encoding the merozoite antigen PfCyRPA-bio-his (Addgene plasmid #50823) was a gift from Gavin Wright (Wellcome Sanger Institute, Cambridge, UK). The vector contained the 3D7 PfCyRPA DNA sequence encoding amino acids D29-E362 with three mutations introduced to remove N-linked glycosylations (S147A, T324A and T340A). Furthermore, the construct included a C-terminal region comprising ratCD4 (d3+4), followed by a biotinylation sequence and a hexahistidine (His6) tag (24 (link)).
Transient transfection of PfCyRPA-bio-his was performed using the Expi293F™ Expression System Kit (Gibco), as per manufacturer’s instructions. Supernatant was harvested five days post transfection for purification. The antigen was purified by IMAC chromatography using a 5 mL HiTrap IMAC HP column charged with 0.1 M NiSO₄ x 6 H₂O on the ÄKTAxpress system (both GE Healthcare). PfCyRPA was eluted in 500 mM imidazole in sodium phosphate buffer pH 7.2 and buffer-exchanged into PBS using an Amicon ultra centrifugal concentrator (Millipore). Quality of antigen was assessed using SDS-PAGE and western blotting using an anti-his-HRP (C-term) antibody (Miltenyi Biotec).

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 Expi293F^TM 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).