Recombinant Expression and Purification of Norovirus P Proteins

The cDNA fragments encoding the P proteins of two clinical GI.3 huNoVs, the Desert Shield virus DSV395 (GenBank code: U04469.1, VP1 amino acids 227–544) and the VA98115/1998 (GenBank code: AY038598.1, VP1 amino acid 227–538), were chemically synthesized and cloned into expression vector pGEX-6P-1 at EcoRI and SalI/XhoI restriction sites. The P proteins were expressed in E. coli BL21(DE3) as described elsewhere (47 (link)– (link)49 (link)). The resulting GST-P fusion proteins were purified using glutathione-Sepharose 4B (GE Healthcare Life Sciences) according to the manufacturer’s instructions, and the GST tag was removed with Prescission protease at 4°C overnight. The P proteins were further purified by anion ion exchange using Mono Q 5/50 GL (GE Healthcare Life Sciences) and gel-filtration chromatography using Superdex75 (GE Healthcare Life Sciences), in a buffer containing 20 mM HEPES pH 7.5 and 150 mM NaCl (Fig. S1A and B). Purified P proteins were concentrated to 8 mg/mL for crystallization. The P protein mutants of VA115 and DSV were constructed and purified using the same methods as for native P proteins.