Recombinant Baculovirus Protein Purification

Recombinant baculoviruses encoding POLγB and the different POLγA versions were expressed in Sf9 cells^{32 (link)}. These recombinant proteins all lacked the N-terminal mitochondrial targeting sequence and carried a carboxy-terminal 6 × His-tag. The proteins were purified over HIS-Select Nickel Affinity Gel (Sigma-Aldrich) and HiTrap Heparin HP (GE Healthcare), followed by HiTrap SP HP or HiTrap Q HP columns (GE Healthcare), depending on the net electrical charge of the protein. MtSSB lacking the N-terminal mitochondrial targeting sequence was expressed in insect cells and purified over DEAE Sepharose Fast Flow (GE Healthcare), HiTrap Heparin HP and HiTrap SP HP, followed by gel filtration using HiLoad Superdex 200 (GE Healthcare).