Ebola virus VP35 has novel NTPase and helicase-like activities

Shu, Ting; Gan, Tianyu; Bai, Peng; Wang, Xiaotong; Qian, Qi; Zhou, Hui; Cheng, Qi; Qiu, Yang; Yin, Lei; Zhong, Jin; Zhou, Xi

The expression and purification of proteins from baculovirus system were performed as previously described (38, 40) . Briefly, Sf9 cells were infected with the recombinant baculoviruses and harvested at 3 days post-infection. Cell pellets were re-suspended, lysed by sonication and subject to centrifugation for 30 min at 11 000 g to remove debris. To get rid of the possible contaminant co-purified from MBP-VP35 via binding to RNA, the supernatant was treated with RNase A (Omega) at the final concentration of 0.1 g/l for 4 h. Then, the protein in the supernatant was purified using amylase affinity chromatography (New England BioLabs, Ipswich, MA, USA) according to the manufacturer's protocol. For His6-fusion protein, the protein in the supernatant was purified by Ni-NTA agarose column (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. All the purified proteins were concentrated using Amicon Ultra-30 filters (Millipore, Schwalbach, Germany). After that, the store buffer was exchanged to 50 mM 2-[4-(2-hydroxyethyl)-1piperazinyl] ethanesulfonic acid (HEPES)-KOH (pH8.0). All proteins were quantified by the Bradford method and stored at -80 • C in aliquots. Proteins were separated on 10% SDS-PAGE and visualized by Coomassie blue.