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 assembly of the viral negative-sense genomic RNA. The Sin Nombre N protein was expressed as a C-terminal hexahistidine fusion in Escherichia coli and initially purified by nickel-affinity chromatography. We developed methods to extract the soluble fraction and to solubilize the remainder of the N protein using denaturants. Maximal expression of protein from native purification was observed after a 1.5-h induction with IPTG (2.4 mg/L). The zwitterionic 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 chromatography, were also subjected to Hi Trap SP Sepharose fast-flow (FF) chromatography. 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 contaminant. The apparent dissociation constant of the N protein, purified by nickel-affinity and SP Sepharose FF chromatography, and the 5' end of the viral S-segment genome were measured using a filter binding assay. The N protein-vRNA complex had an apparent dissociation constant of 140 nM.
Jonsson
C B
CB
Department of Chemistry, New Mexico State University, Las Cruces 88003, USA. cjonsson@nmsu.edu
Gallegos
J
J
Ferro
P
P
Severson
W
W
Xu
X
X
Schmaljohn
C S
CS
Fero
P
P
D000345:Affinity Labels; D002793:Cholic Acids / Q000494:pharmacology; D002845:Chromatography; D002846:Chromatography, Affinity; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000821:virology*; D006639:Histidine; D019590:Nucleocapsid Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D029261:Sin Nombre virus / Q000737:chemistry*
D000345:Affinity Labels; D002793:Cholic Acids; D003902:Detergents; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins; D006639:Histidine; D012313:RNA; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate