Recombinant PfCSP Expression and Purification

Recombinant PfCSP constructs were produced as previously described.^{8 (link)} Briefly, sequences corresponding to rPfCSPFL and Junctional region (rPfCSP5/3) were cloned into the same CMV/R-expression vectors with a C-terminal AviTag, HRV3C-processing tag, and a 6X histidine tag (GenScript). rPfCSP constructs were expressed through transient transfection in 293F cells using the Freestyle 293F expression system (Thermo Fisher Scientific) at 37°C, 8% CO₂ for 6 days, and purified from culture supernatants through polyhistidine-tag affinity chromatography followed by size exclusion chromatography (SEC) on an ÄKTA^™ Start (GE Healthcare). Monomer-containing fractions were pooled, concentrated, snap frozen, and stored at −80°C. Peptides 21, 22, and 29 were produced by direct synthesis and biotinylated by GenScript.