The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension

te Velthuis, Aartjan J.W.; van den Worm, Sjoerd H. E.; Snijder, Eric J.

For SARS-CoV nsp7-nsp8 expression, the sequence encoding amino acids 3837-4117 of the SARS-CoV replicase pp1a was amplified by reverse transcriptionpolymerase chain reaction (RT-PCR) from the genome of SARS-CoV isolate Frankfurt-1 (Genbank accession number AY291315). The primers used were SAV704 and SAV429 (Supplementary Table S1 ). For nsp8 expression, the sequence encoding pp1a residues 3920 to 4117 was amplified by RT-PCR using SAV428 and SAV429 as primers (Supplementary Table S1 ). Both PCR products were digested with SacII and BamHI, and ligated into expression vector pASK3-Ub-CHis 6 (10). This vector was originally derived from the pET26-Ub-CHis 6 vector (14) , but drives expression of N-terminally ubiquitintagged and C-terminally His 6 -tagged fusion proteins via a tetracyclin-inducible promoter, to rule out the potential T7 polymerase contaminations that are known to cause false positive results when using T7 promoter-driven systems for recombinant RdRp expression. All described nsp8 mutants were engineered via site-directed mutagenesis according to the QuikChange protocol (Stratagene) using the primers listed in Supplementary Table S2. For nsp7-8 or nsp8 expression, Escherichia coli C2523 cells (New England Biolabs) were transformed with the plasmids pASK3-Ub-nsp7-8-CHis 6 or pASK3-Ub-nsp8-CHis 6 together with the Ubp1 protease expression plasmid pCG1 (14) . Routinely, 50 ml of Luria Broth, containing ampicillin (50 mg/ml) and chloramphenicol (34 mg/ml), was inoculated 1:1000 with o/n precultures, and cells were grown to OD 600 >0.8 at 37 C. Subsequently, the cells were slowly cooled to 20 C, followed by induction with anhydrotetracycline (Fluka) at a final concentration of 200 ng/ml for 16 h. Expression at 20 C was, however, only crucial for the preparation of certain nsp8 mutants and similar yields of active wild-type protein could be obtained by expression at 37 C for 3-4 h. Cells were harvested by centrifugation and stored at 20 C until protein purification was started.