Expi293 transient expression system

The recombinant E (rE) proteins of DENV1 (aa 1–397), DENV2 (aa 1–395), DENV3 (aa 1–395) and DENV4 (aa 1–397) were expressed by the EXPI293 transient expression system (ThermoFisher) following supplied protocols. The previously described expression construct was used [15 ] where an N-terminal IL2 secretion peptide leads the homotypic prM-rE cassette for each serotype. All rE proteins were equipped with a C-terminal 6×His-tag (SSGGSHHHHHH). Protein expression was driven by a CMV early enhancer β-actin promoter.
Recombinant proteins were purified as previously described [15 ]. In short, expression supernatants were concentrated and buffer exchanged by tangential flow filtration and proteins were purified by Ni²⁺-affinity chromatography. Elution fractions containing the rE-proteins were pooled and subjected to size-exclusion chromatography. Finally, the purified and concentrated proteins were flash frozen and stored at -80°C until further use.

The soluble recombinant DENV2 envelope protein (sRecE, aa1-395) was expressed using the EXPI293 transient expression system (ThermoFisher) using manufacturer supplied protocols. The N-terminal IL2 secretion leader peptide was fused to the DENV2 prM-sRecE cassette. sRecE was equipped with a C-terminal 6x histidine purification tag sequence (SSGGSHHHHHH) and expression was driven by a CAG (CMV early enhancer β actin) promoter. Supernatants were concentrated and buffer exchanged into a Ni⁺² binding buffer (50mM NaPO₄, 500mM NaCl, 25mM imidazole, 0.02% Na-Azide) via a tangential flow filtration and subjected to Ni⁺² affinity chromatography. After washing with Ni⁺² binding buffer, the Ni⁺² column was step eluted with elution buffer (50mM NaPO₄, 500mM NaCl, 500mM Imidazole 0.02%, Na-Azide, 10% glycerol). The fractions containing envelope protein were pooled and concentrated before being subjected to size exclusion chromatography using a 16/60 Superdex S200 column that was equilibrated with PBS containing 10% glycerol. Fractions with envelope protein were pooled, concentrated to 3mg/ml, flash frozen in liquid N₂, and stored at -80°C. The resulting envelope protein was >95% pure as assessed by SDS-PAGE.

The soluble recombinant DENV2 (sRecE, aa 1–395) and ZIKV (aa 1–404) envelope proteins were expressed by the EXPI293 transient expression system (ThermoFisher) as previously described^{24 (link)}. In short: DENV2 and ZIKV sRecE was equipped with a C-terminal 6x His tag and expression was driven by a CAG promoter. Through tangential flow filtration, supernatants were concentrated and buffer exchanged into a Ni²⁺ binding buffer (50 mM NaPO₄, 500 mM NaCl, 25 mM imidazole, 0.02% Na-Azide) and subsequently Ni²⁺ affinity purified. After washing, the Ni²⁺ column was serially eluted with elution buffer (50 mM NaPO₄, 500 mM NaCl, 500 mM Imidazole 0.02%, Na-Azide, 10% glycerol). Fractions containing envelope proteins were pooled subjected to size exclusion chromatography using a 16/60 Superdex S200^TM column. Fractions with envelope protein were pooled in PBS + 10% glycerol, concentrated and flash frozen in liquid N₂, and stored at −80 °C. The oligomeric state of the purified proteins were analyzed size exclusion chromatography using a Superdex S200^TM column connected to multi angle light scattering instrument (Wyatt DAWN HELEOS-II) with an OptiLab T-rex refractometer. 200 µg of DENV2 sRecE was analyzed with or without 40 µg of 2D22 in 100 µl PBS.