S200 increase column

The coding sequence for the entire prM and soluble E (sE) regions (amino acid residues 122–678 of the YFV polyprotein) of the YFV Asibi Strain (Uniprot ID: Q6DV88) or YFV ES-504 strain (Uniprot ID: A0A1W6I1A1) were cloned into an insect expression vector encoding a C-terminal double strep tag, pMT-puro. The expression construct design was based on previously published structures of flavivirus antigens (Modis et al., 2003 (link), 2005 (link); Rey et al., 1995 (link)). The YFV prM/E constructs were used to generate an inducible, stable Drosophila S2 line. Protein expression was induced with addition of copper sulfate and allowed to proceed for 5 to 7 days. Recombinant proteins were affinity purified from the culture supernatant with a StrepTrap HP column (GE Healthcare) and an additional purification step was carried out using size-exclusion chromatography step using an S200 Increase column (GE Healthcare). The final protein preparations were stored in PBS (pH 7.4) supplemented with an additional 150 mM NaCl, aliquoted and stored at −70°C.

To determine the epitope of HR2/MPER-directed mAbs, BDBV223, 317, or 340 Fabs were generated as described above and added in 10 M excess to BDBV GPΔmuc and allowed to bind overnight at 4°C^{9 (link)}. Complexes were subsequently purified by size exclusion chromatography on an S200 Increase column (GE) and stained as previously described^{7 (link)}. Particles were visualized using an FEI Tecnai Spirit electron microscope operating at 120kV and images were collected on a TVIPS TemCam-F416 (4k x 4k) CCD camera using Leginon^{32 (link)} with the following settings: magnification of 52,000X that resulted in a pixel size of 2.05Å at the specimen plane, a constant defocus of -1.00 μm and an electron dosage of ~30e⁻/Ų. Images were processed using the Appion platform^{33 (link)}. Particles were picked using DoG Picker^{34 (link)}, stacks were created and 2D reference-free class averages were generated using iterative MRA-MSA^{35 (link)}. For all complexes, there was a strong bias toward side-views. Further, the region containing the HR2/MPER epitope in our soluble GP constructs is flexible, as indicated by the variety of positions that bound Fabs adopted in the class averages. Therefore, our data was refractory to a reconstruction, although class averages could be compared to previous class averages of known complexes to determine the spatial location of the epitope on GP.

Purified arrestin-3-(1–393) and its loop insertion mutant were incubated with or without 100 μM of IP₆ for 30 min and then injected onto a S-200 Increase column (GE Healthcare) equilibrated with 20 mM MOPS (pH7.5), 150 mM NaCl and 1 mM TCEP. The retention volume was used to estimate the average molecular weight using a standard curve.