The regions corresponding to the entire extracellular domains of 51 merozoite cell-surface proteins from the P. falciparum 3D7 strain were determined by using transmembrane and GPI-anchor (33 (link), 34 (link)), or signal peptide (35 (link)) prediction software. Sequences encoding the extracellular domains of these proteins, with the exception of their signal peptide, were made by gene synthesis (GeneartAG) and are presented in Table I. All sequences were codon-optimized for expression in human cells and all potential N-linked glycosylation sites (NXS/T) were modified by substituting the serine or threonine residue with an alanine residue to prevent the inappropriate addition of large glycans that are absent in the native P. falciparum proteins. The coding sequences were flanked by unique NotI and AscI sites and cloned into a derivative of the pTT3 expression vector (36 (link)) between the leader sequence of the mouse variable κ light chain 7–33 (37 (link)), and a rat Cd4 domains 3 and 4 tag followed by an enzymatic biotinylation sequence as previously described (38 (link)). All expression constructs were cotransfected with the BirA biotinylation enzyme into HEK293E cells and are available from Addgene, a non-profit plasmid repository (www.addgene.org). The soluble biotinylated recombinant proteins were collected from the cell culture supernatant 6 days post-transfection, and dialyzed into HBS before analysis. During gene synthesis, constructs encoding the full-length ectodomain of RH1, RH2b and RH4 proved to be toxic in bacteria, and only subfragments could be produced as presented in Table I.