Journal: bioRxiv

Article Title: Molecular architecture and domain arrangement of the placental malaria protein VAR2CSA suggests a model for receptor binding

doi: 10.1101/2020.04.16.045096

Figure Lengend Snippet: VAR2CSA and its deletion constructs expressed in HEK293-F cells and SDS-PAGE of the purified recombinant VAR2CSA proteins. (A) Schematic indicating the locations of DBL domains for constructs I-V. For each construct, the numbers at either end denote the beginning and ending amino acid sequence numbers. I, II and IV contain a non-cleavable C-terminal cMyc tag and hexahistidine tag, as coded in the commercial vector. For the III and V constructs, the C-terminal tag is replaced with a TEV-cleavable 3X-FLAG tag and a hexahistidine tag, as defined in Table S2, GBLOCK2. In addition, P1 contains a glycine and threonine residue inserted between native residues 58 and 59 as a cloning artefact. (B) Purified proteins (each 2 μg/lane), electrophoresed using 4-20 % gradient gels (1 mm thick) under reducing conditions and (C) non-reducing conditions are shown. In each gel, the lanes are: M, Molecular weight markers; I, NTS-DBL6ε (~310 kDa); II, DBL1x-ID2a (~125 kDa); III, ID2b-DBL6ε (~200 kDa); IV, DBL3x-DBL6ε (~180 kDa); V, DBL4ε-DBL6ε (~130 kDa). The molecular mass marker (kDa) standards are indicated in the left margin.

Article Snippet: cMyc mAb (Cat NB600-302, Novus Biologicals, CO) and NTS-DBL6ε or DBL1x-ID2a proteins were mixed at a ratio of 1:2 and incubated for 30 min on ice.

Techniques: Construct, SDS Page, Purification, Recombinant, Sequencing, Plasmid Preparation, FLAG-tag, Residue, Cloning, Molecular Weight, Marker

Journal: bioRxiv

Article Title: Molecular architecture and domain arrangement of the placental malaria protein VAR2CSA suggests a model for receptor binding

doi: 10.1101/2020.04.16.045096

Figure Lengend Snippet: VAR2CSA is an asymmetric molecule with the C-terminus located within the smaller lobe. (A) Micrograph of NTS-DBL6ε. The white bar is 50 nm. (B) Subset of 2D classes obtained during initial 2D classification of NTS-DBL6ε. (C) Single 2D class showing the layers (L1, L2, L3), separated by broken white lines, defined in the text. (D) Micrograph of NTS-DBL6ε bound to anti-cMyc antibody. (E) Representative 2D classes of unbound NTS-DBL6ε. (F) Representative 2D classes of unbound anti-cMyc antibody. (G) Representative 2D classes of NTS-DBL6ε:anti-cMyc-antibody complex. The letter A indicates the location of the cMyc antibody at the tip of the smaller lobe of NTS-DBL6ε.

Article Snippet: cMyc mAb (Cat NB600-302, Novus Biologicals, CO) and NTS-DBL6ε or DBL1x-ID2a proteins were mixed at a ratio of 1:2 and incubated for 30 min on ice.

Techniques: