Mechanism of Nucleic Acid Unwinding by SARS-CoV Helicase

Adedeji, Adeyemi O.; Marchand, Bruno; te Velthuis, Aartjan J. W.; Snijder, Eric J.; Weiss, Susan; Eoff, Robert L.; Singh, Kamalendra; Sarafianos, Stefan G.

C-terminal hexahistidine containing nsp12-(nsp12-H 6 ) was cloned, expressed and purified as described previously [61, 62] . Briefly nsp12 was PCR amplified using 59SacII nsp12-GCGGGTACCCC GCGGTGGATCTGCGGATGCATCAA-39 and 59BamHI nsp13-ATGCTAGGATCCGCTGTAGG TGCTTGTG (forward primer), and 59BamHI nsp13-GCGCGATCGGGATCCCTGCAAGACT GTATGT (reverse primer). The PCR amplicon was digested with SacII and BamHI, and ligated into expression vector pET26-Ub-CHis 6 followed by subcloning of the ubiquitin-nsp12-His 6 fusion gene (Ub-nsp12-CHis 6 ) into vector pASK3 (IBA). Protein expression and purification was performed as described previously using Talon beads [35] . The protein was eluted with 20 mM HEPES pH 7.4, 0.5 M NaCl, 200 mM imidazole, 0.1% Triton X-100 and further purified on a Superdex75 10/300GL column (20 mM HEPES pH 7.4, 100 mM NaCl, 1 mM DTT, 0.1% Triton-X 100, and 5% glycerol). Fractions containing the desired protein were concentrated and stored at 280uC.