Purification and characterization of the sin nombre virus nucleocapsid protein expressed in Escherichia coli

Sin Nombre virus is a member of the Hantavirus genus, family Bunyaviridae, and is an etiologic agent of hantavirus pulmonary syndrome. The hantavirus nucleocapsid (N) protein plays an important role in the encapsidation and a ssembly of the viral negative-sense genomic RNA. The Sin Nombre N protein w as expressed as a C-terminal hexahistidine fusion in Escherichia coli and i nitially purified by nickel-affinity chromatography. We developed methods t o extract the soluble fraction and to solubilize the remainder of the N pro tein using denaturants. Maximal expression of protein from native purificat ion was observed after a 1.5-h induction with IPTG (2.4 mg/L). The zwitteri onic detergent Chaps did not enhance the yield of native purifications, but increased the yield of protein obtained from insoluble purifications. Both soluble and insoluble materials, purified by nickel-affinity chromatograph y, were also subjected to Hi Trap SP Sepharose fast-flow (FF) chromatograph y. Both soluble and insoluble proteins had a similar A(280) profile on the Sepharose FF column, and both suggested the presence of a nucleic acid cont aminant. The apparent dissociation constant of the N protein, purified by n ickel-affinity and SP Sepharose FF chromatography, and the 5' end of the vi ral S-segment genome were measured using a filter binding assay. The N prot ein-vRNA complex had an apparent dissociation constant of 140 nM. (C) 2001 Academic Press.