Figure 1.

Schematic representation of 4.1R and EBA-181 and purification of the respective fusion proteins. (A) Schematic of 4.1 cDNA showing alternative and constitutive exons. The erythrocyte translation initiation site is indicated at nucleotide (nt) position 816 in exon 4 with the coding sequence extending to position 2737. The corresponding full-length 80 kDa-4.1R molecule is shown below the cDNA. Amino acid (aa) residue numbers depict the relative locations of the four 4.1R structural domains [26-28]. The 30 kDa domain (aa 1–297) is located at the N-terminal end of the protein and corresponds to nt 816–1716. The 16 kDa and 10 kDa domains are encoded by nt 1717–2285 (aa 298–471), with the 22 kDa region situated at the C-terminal end. (B) GST-4.1R fusion domains were expressed and purified using glutathione magnetic affinity beads. Approximately 1–2 μg of total protein was resolved by 12% SDS-PAGE. The protein samples are erythrocyte membrane marker M (lane 1), GST control (lane 2), GST-10 kDa (lane 3), GST-16 kDa (lane 4), GST-22 kDa (lane 5) and GST-30 kDa (lane 6). (C) Schematic of the full-length P. falciparum invasion protein EBA-181 [43], accession number: PFA0125c. Amino acid (aa) numbers underneath the schematic demarcate the various domains in the protein. The molecule comprises two DBL domains (denoted F1 and F2) which define erythrocyte specificity, as well as two transmembrane regions and a C-terminal cysteine-rich motif. The relative position of the EBA-181 fragment (PfJ) used in this study is shown (aa 945–1097; expected size 25 kDa). (D) 6His-PfJ was expressed and purified from crude E. coli lysates using magnetic nickel beads and resolved by SDS-PAGE: M, erythrocyte membrane marker (lane 1) and purified PfJ (lane 2), which resolves at ~50 kDa.