Recombinant PfRH5 Protein Production

All chemicals were purchased from Sigma–Aldrich, UK unless otherwise specified. The design of the PfRH5 protein with C-terminal His6 tag has been described elsewhere, where it was reported as variant version 1.0 (Hjerrild et al., 2016 (link)). In brief, the protein encodes the full-length ectodomain of the PfRH5 antigen (aa E26-Q526) based on the sequence of the 7G8 laboratory-adapted P. falciparum parasite line, and all four putative N-linked glycosylation sequons (N-X-S/T) were mutated Thr to Ala – as performed for a previous PfRH5 protein vaccine produced in mammalian HEK293 cells and tested in rabbits (Bustamante et al., 2013 (link)) and Aotus monkeys (Douglas et al., 2015 (link)). A synthetic gene was designed based on the above 7G8 sequence for PfRH5 and codon-optimised for expression in D. melanogaster (GeneArt, Thermo Fisher Scientific, Germany). The construct also contained a Kozak sequence (GCC ACC) at the 5′ end, an N-terminal 18 aa Ig heavy chain binding protein (BiP) insect signal peptide (MKLCILLAVVAFVGLSLG) and a C-terminal His6 tag. This gene insert was subcloned into the pExpreS²-1 plasmid allowing for Zeocin selection (ExpreS²ion Biotechnologies, Denmark). Subsequently, the C-terminal His6 tag coding sequence was replaced within the plasmid with that encoding the C-tag – four amino acids E-P-E-A.