Baculovirus-Mediated Expression and Purification of APOBEC3H Variants

The pFAST-bac1 vectors were used to produce recombinant baculovirus according to the protocol for the Bac-to-Bac system (Life Technologies). Expression and purification of GST-A3H Hap II and GST-A3H Hap VII were previously described (39 (link),67 (link)). Recombinant GST-A3H Hap I WT and mutant baculovirus (K117E, K117E/K121E) were used to infect Sf9 cells at a multiplicity of infection of 1 and cells were harvested after 72 h. Cells lysates were treated with 100 μg ml⁻¹ of RNase A (Qiagen). Lysates were cleared by centrifugation and then incubated with Glutathione Sepharose 4B resin (GE Healthcare) at 4°C overnight and subjected to a series of salt washes (0.25–1 M NaCl) as described previously (68 (link)). On-column cleavage from the GST tag with Thrombin (GE Healthcare) was performed at 21°C for 18 h in Thrombin digestion buffer [20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol and 2 mM dithiothreitol (DTT)]. Proteins were assessed to be 90% pure by SDS-PAGE (Supplementary Figure S1).