Histrap hp ni sepharose column

Lysates were purified using a
1 mL HisTrap HP Ni-sepharose column (GE Healthcare Life Sciences)
connected to an ÄKTA pure protein purification system (Cytiva).
The column was equilibrated with 5 CV Buffer A (50 mM Tris-HCl, 300
mM NaCl, pH 8.0) before the supernatant was loaded onto the column.
Impurities were removed by washing with Buffer A for 10 CV. His-tagged
proteins were eluted using a gradient of 0–100% Buffer B (50
mM Tris-HCl, 300 mM NaCl, 400 mM imidazole, pH 8.0) over 40 CV. All
steps were performed using a flow rate of 1 mL/min. GFP-containing
fractions were identified using 488 nm absorbance.

The sequence encoding amino acid residues (aa) 384–661 of HCV genotype 1a strain H77c (GenBank accession no. AF009606), plus an N-terminal 6-histidine tag, was inserted into the pAcGP67-A transfer vector (BD Biosciences). Sf9 cells were co-transfected with the transfer vector and Baculogold™ linearized baculovirus DNA, to make recombinant baculovirus (rBac) expressing the ectodomain of E2 as a secreted, soluble protein. The rBac E2₆₆₁ was cloned by limiting dilution, amplified, and used to infect Hi Five™ cells at a multiplicity of infection (moi) of 1. After 3 days’ incubation the supernatant medium was harvested, filtered through a 0.22 µm membrane, and dialyzed first against 20 mM sodium phosphate pH 6.8, 150 mM NaCl and subsequently against 20 mM sodium phosphate pH 8.0, 300 mM NaCl. Ultrapure imidazole was added to a concentration of 20 mM, and the supernatant loaded onto a HisTrap HP Ni Sepharose column using an Äkta Purifier (GE Healthcare). E2₆₆₁ was eluted with a gradient of 40–200 mM imidazole in 20 mM sodium phosphate pH 8.0, 300 mM NaCl, to separate monomeric molecules from dimers and multimers. Monomeric E2₆₆₁ was purified to homogeneity by size exclusion chromatography (SEC) on a Superdex 200 Increase 10/300 GL column (GE Healthcare).