Hitrap nhs activated hp sepharose column

The plant‐derived E2 antigen was produced in N. benthamiana plants. After 7–8 days post infiltration (DPI), plants were harvested and homogenized in two volumes (w:v) of cold buffer (50 mm sodium phosphate, 150 mm sodium chloride, 70 mm ascorbic acid, 5 mm EDTA, pH 8.0) and centrifuged at 14 000 g for 15 min at 12 °C. Major host cell proteins were precipitated by adjusting the pH of the extract to 4.8 with 2 m acetic acid. After a 2‐min incubation, the pH of the extract was adjusted to pH 7–8. The extract was centrifuged again at 14 000 g for 15 min at 12 °C and clarified through a 0.2 μm Sartopure PP3, size 4 filter (Sartorius, Bohemia, NY). For E2‐StrepII antigen purification, extracts were loaded on 5 mL StrepTrap HP column (GE HealthCare Life Sciences, Piscataway, NJ) following the manufacturer's instructions. For untagged E2 antigen purification, clarified extract was loaded on a HiTrap NHS‐Activated HP Sepharose column (GE HealthCare Life Sciences, Piscataway, NJ) coupled with the anti‐E2 antibody WH303 (APHA, Addlestone, UK). Plant‐made E2 was eluted from the immunoaffinity column with 5 CV of 100 mm citric acid. Elution fractions were then dialysed against 1X Phosphate‐buffered saline (PBS), pH 7.4 overnight at 4 °C. The insect cell‐derived E2 was produced and purified as described previously (Madera et al., 2016).

Soluble PC protein was produced using HEK 293-6E cells (NRC-BRI, Montreal, Canada) following co-transfection of five plasmids encoding the individual PC subunit genes within a pTT5 vector (NCR-BRI). HCMV gH was truncated at 714 amino acids, adding a C-terminal 6xHis tag. The signal peptide from each subunit was replaced with HIV gp120 signal peptide [29 (link)]. Cells were transfected according to manufacturer protocol [30 (link)] and supernatant collected 120 hours later. Secreted PC was captured on Ni²⁺-NTA resin (GE Healthcare, Pittsburgh, PA) and the eluate was further purified using a proprietary monoclonal antibody recognizing a conformational epitope on UL128/130/131A (12E2, [21 (link)]) bound to a HiTrap NHS-Activated HP sepharose column (GE Healthcare). After capturing, PC protein was eluted with 3M MgCl₂ then immediately performed a buffer exchange to 20 mM Tris pH 8.0, 150 mM NaCl followed by gel filtration using a GE Superdex 200 10/300G column. Protein concentration was assessed via Bradford assay and presence of all five subunits was verified via SDS-PAGE followed by Coomassie staining analysis.