Mouse LSECtin as a model for a human Ebola virus receptor

Pipirou, Zoi; Powlesland, Alex S; Steffen, Imke; Pöhlmann, Stefan; Taylor, Maureen E; Drickamer, Kurt

For characterization of the binding specificity of mouse LSECtin by glycan array analysis, a tetrameric form of the CRD was created by attaching a C-terminal biotinylation sequence to the CRD and co-expressing the CRD and biotin ligase ( Figure 2A ). The resulting protein was purified by affinity chromatography as for the untagged protein. Incubation of an excess of biotin-tagged CRD with fluorescently labeled streptavidin resulted in formation of tetramers that could be purified away from uncomplexed CRDs by re-chromatography on a fucose-Sepharose. The tetrameric protein is efficiently retained on a 1-mL affinity column, whereas uncomplexed CRD washes through. The bound CRD-streptavidin complex was then eluted from the affinity column with ethylenediaminetetraacetic acid (EDTA) ( Figure 2B ).