Various var2CSA recombinant proteins expressed in HEK293 cells and <i>E. coli</i>.

<p>(A). Schematic view of the var2CSA domain organization and sequence limits of the recombinant domains studied (3D7-DBL1X, 3D7-DBL2X, FCR3-DBL3X, 3D7-DBL5ε, 3D7-DBL1X-2X, FCR3-DBL3X-4ε, 3D7-DBL1X-CIDR, 3D7-DBL1X-3X, 3D7-DBL1X-4ε, 3D7-DBL1X-5ε and 3D7-DBL1X-6ε). Var2CSA comprises six DBL domains (DBL1X to DBL6ε), a CIDRpam domain and three inter-domain regions (INT1-3) in the extracellular region, together with a trans-membrane segment and acidic C terminus sequence (ATS). DBL1X, DBL2X, DBL3X, DBL4ε, DBL5ε and DBL6ε are shown in green; CIDR in orange; N-terminal sequence (NTS) and inter-domain regions (INT) in grey; the trans-membrane and ATS regions in blue. The length of each bar corresponds to the domain size. (B). Purification of var2CSA derived proteins expressed in HEK293 and <i>E. coli</i>. Nu-SDS-PAGE Precast 4–12% Bis-Tris gel under nonreducing (B) and reducing (C) conditions was loaded with purified recombinant proteins. Lane 1: Marker, lane 2: 3D7-DBL1X, lane 3: 3D7-DBL2X, lane 4: FCR3-DBL3X, lane 5: 3D7-DBL5ε, lane 6: 3D7-DBL1X-2X, lane 7: FCR3-DBL3X-4ε, lane 8: 3D7-DBL1X-CIDR, lane 9: 3D7-DBL1X-3X, lane 10: 3D7-DBL1X-4ε, lane 11: 3D7-DBL1X-5ε and lane 12: 3D7-DBL1X-6ε. Proteins were visualized with Coomassie blue.</p

Depletion of purified adhesion-inhibitory rabbit IgGs (anti 3D7-DBL1X-6ε) by various recombinant proteins.

<p>Immunization of rabbit with 3D7-DBL1X-6ε induces adhesion-blocking antibodies. Purified IgG against 3D7-DBL1X-6ε (Anti-DBL1X-6ε was depleted on 3D7-DBL1X, 3D7-DBL2X, FCR3-DBL3X, 3D7-DBL5ε, 3D7-DBL6ε, 3D7-DBL1X-2X, 3D7-DBL1X-CIDR, FCR3-DBL3X-4ε, 3D7-DBL1X-3X, 3D7-DBL1X-4ε, 3D7-DBL1X-5ε and 3D7-DBL1X-6ε immobilized on tosylactivated beads. Unbound (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020270#pone-0020270-g005" target="_blank">Fig. 5A</a>) and bound (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020270#pone-0020270-g005" target="_blank">Fig. 5B</a>) fractions were tested for inhibition of binding of 3D7-DBL1X-6ε to decorin. Data are express as % of inhibition compare to control.</p

Competitive inhibition of recombinant DBL domains binding to decorin using various glycosaminoglycans.

<p>Recombinant proteins (A) 3D7-DBL1X-2X, (B) 3D7-DBL1X-CIDR, (C) 3D7-DBL1X-3X, (D) 3D7-DBL1X-4ε, (E) 3D7-DBL1X-5ε and (E) 3D7-DBL1X-6ε at 1 µg/mL were premixed with increasing amounts of BSA (□) or glycosaminoglycans, 0.156–100 µg/mL of CSA (×), CSC (⋄) or HS (▵) and incubated in plates previously coated with decorin.</p

K_D for var2CSA recombinant proteins binding to placental CSPG.

<p>K_D values were determined from the concentration dependence of steady-state SPR response using the Biacore BIAEVALUATION 3.1 software.</p><p>*: from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020270#pone.0020270-Srivastava1" target="_blank">[34]</a>.</p>#<p>: from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020270#pone.0020270-Gangnard1" target="_blank">[41]</a>.</p

Binding of recombinant DBL domains from 3D7 and FCR3-DBL1X-6ε to different glycosaminoglycans.

<p>ELISA-based direct binding assay was performed to identify the specificity of (A) 3D7-DBL2X, (B) FCR3-DBL3X, (C) 3D7-DBL5ε, (D) FCR3-DBL3X-4ε, (E) 3D7-DBL1X-2X, (F) 3D7-DBL1X-CIDR, (G) 3D7-DBL1X-3X, (H) 3D7-DBL1X-4ε, (I) 3D7-DBL1X-5ε and (J) 3D7-DBL1X-6ε to different sulfated glycosaminoglycans. Increasing concentrations of recombinant proteins at serial dilutions of 0.31–20 µg/mL were added to wells previously coated with BSA (▵) or different glycosaminoglycans: decorin (○), CSA (□), CSC (⋄), HS (×).</p