Crystal structure of Middle East respiratory syndrome coronavirus helicase

Hao, Wei; Wojdyla, Justyna Aleksandra; Zhao, Rong; Han, Ruiyun; Das, Rajat; Zlatev, Ivan; Manoharan, Muthiah; Wang, Meitian; Cui, Sheng

The gene encoding full-length MERS-CoV nsp13 helicase (1-598aa, GeneBank accession: YP_009047202) was amplified by polymerase chain reaction (PCR) and inserted via BamHI/ XhoI restriction sites into a modified pFastbac-1 baculovirus transfer vector with an N-terminal 6×His-SUMO tag [38] . Nsp13 protein was overexpressed in High-5 insect cells using Bacto-Bac Baculovirus Expression System (Invitrogen). One liter cell culture (2.0×10 6 cells ml -1 ) was infected with 30 ml baculovirus at 22˚C. Forty-eight hours after infection, cells were harvested by centrifugation. The cell pellet was re-suspended in a lysis buffer containing 25mM Tris-HCl (pH 7.5), 1.5 M NaCl and 15mM imidazole and lysed by ultrasonification. High salt concentration in the lysis buffer was necessary to remove nucleic acids bound to nsp13. Protein was purified using Ni-NTA resin (QIAGEN). The eluted protein was digested with Pre-Scission protease (GE healthcare) to remove the 6×His-SUMO tag. Finally, the untagged nsp13 was purified using size-exclusion chromatography (Superdex-200, GE healthcare). The purified nsp13 was concentrated to~8 mg/ml in the buffer containing 10mM HEPES (pH 7.0) and 100mM NaCl. Before crystallization trials, nsp13 was mixed with 5'-triphosphate 15 thymine single-stranded DNA (ppp-15T) with 1:1.5 molar ratio and incubated at 4˚C overnight. Crystallization of nsp13 was achieved by mixing equal volume of sample and reservoir buffer (1.0μl) containing 0.1 M Tris-HCl (pH 8.5), 1M (NH 4 ) 2 SO 4 and 15% glycerol. The crystals were grown in a hanging-drop vapor-diffusion system at 18˚C.