Crystal Structure of the C-Terminal Cytoplasmic Domain of Non-Structural Protein 4 from Mouse Hepatitis Virus A59

Xu, Xiaoling; Lou, Zhiyong; Ma, Yanlin; Chen, Xuehui; Yang, Zhangsheng; Tong, Xiaohang; Zhao, Qi; Xu, Yuanyuan; Deng, Hongyu; Bartlam, Mark; Rao, Zihe

Using a multiple sequence alignment of nsp4 from representatives of the genus Coronaviridae, together with TMHMM and Tmpred predictions from the primary sequence, we successfully designed the construct of this highly conserved hydrophilic domain of MHV-A59 nsp4. The gene fragment encoding the MHV-A59 nsp4 carboxy terminal domain (nsp4C) spanning T3252 to Q3328 of pp1a was cloned from the virus genomic cDNA library by polymerase chain reaction (PCR) into the pGEX-6p-1 vector (GE Healthcare), and the resulting plasmid was transformed into Escherichia coli BL21 (DE3) cells. The recombinant glutathione transferase (GST) fusion protein, GST2nsp4C, was purified by GST-glutathione affinity chromatography. The cells were disrupted by sonication in 50 mM Tris-Cl pH 8.5, 200 mM NaCl and 2 mM DTT, and the cell lysates were centrifuged at 15,000 rpm for 30 min. The soluble fraction was then applied on a GSTaffinity chromatography column for purification. The GST tag was removed by PreScission protease (GE Healthcare), leading to five additional residues (GPLGS) at the N-terminus. The protein sample purified by affinity chromatography was further purified by anion ion-exchange chromatography on a ResourceQ column with an elution buffer containing 50 mM Tris-Cl, pH 8.5 and a gradient concentration of NaCl to 1 M. The flow through fraction from Resource Q column was collected for gel filtration purification on a Superdex75 column in buffer containing 50 mM Tris-Cl pH 8.5 and 150 mM NaCl. A C425S site-directed mutant was constructed by overlapping extension PCR [40] , and was then expressed, and purified using the same protocol.