Producing Hepatitis B Virus Capsids

HBV Cp was expressed in E. coli BL21(DE3) cells (T7 Expression strain, New England Biolabs), expressed from a pET28b plasmid. Induction with 1 mM isopropyl-β-D-thiogalactoside (IPTG) at an optical density (OD₆₀₀) of ~0.6 was followed by growth for 20 h at 21 °C. Cells were lysed using a Soniprep 150 and clarified by spinning at 11,000 × g for 1 h. NCPs were then pelleted by centrifugation at 120,000 × g for 14 h. Pellets were resuspended in 20 mM HEPES (pH 7.5), 250 mM NaCl, 5 mM dithiothreitol (DTT) and applied to a prepacked Captocore 700 column (GE Life Sciences). Fractions containing NCPs were pooled and precipitated with 40% (w/v) ammonium sulfate. NCPs were dissociated into Cp dimers by dialysis into 1.5 M guanidinium chloride (GuHCl) as previously described^{12 (link),38 (link)}. All steps after sonication were performed in the presence of complete protease inhibitor tablets (1 tablet per 500 mL buffer, Thermofisher Scientific). Cp dimer concentration was determined by the absorbance at 280 nm (ε₂₈₀ of Cp₂ = 55,920 L mol⁻¹ cm⁻¹). Cp fractions with an A_260/280 ratio of ≤0.65 were used in assembly assays.