Construction, expression, production and purification of recombinant proteins

Recombinant P. falciparum CSP (3D7) and CSP fused with human MIP-3α proteins were prepared as previously described¹⁶. The codon-optimized CSP DNA sequence containing deletions of the N-terminal 20 aa signaling sequence and the C-terminal 23 aa anchor region fused with human MIP-3α was used to express MIP-3α-CSP chimeric protein (MCSP). Twenty-two NANP repeats were included in the truncated CSP. CSP and MCSP sequences were cloned into pET-47b (Novagen Inc., Madison, WI) fused with 6XHis. The proteins were expressed in BL21 DE3 competent cells (New England Biolabs, Ipswich, MA). Protein purification was undertaken using nickel-affinity chromatography (Qiagen, Valencia, CA, USA) and endotoxin was removed by two-phase extraction with Triton X-114²². Protein concentration was measured by Bradford assay (BioRad, Hercules, CA). Endotoxin concentration was determined using the ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit (Genscript, NJ). All protein used for immunization had final endotoxin levels below 10 EU/ml. Polyacrylamide gel electrophoresis of the final products are shown in Fig. S1.