Purification and characterization of the Sin Nombre virus nucleocapsid protein expressed in Escherichia coli

Colleen Jonsson, Juan Gallegos, Phillip Fero, William Severson, Xiaolin Xu, Connie S. Schmaljohn

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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 A280 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 proteinvRNA complex had an apparent dissociation constant of 140 nM.

Original languageEnglish (US)
Pages (from-to)134-141
Number of pages8
JournalProtein Expression and Purification
Volume23
Issue number1
DOIs
StatePublished - Jan 1 2001

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Sin Nombre virus
Nucleocapsid Proteins
Escherichia coli
Nickel
Hantavirus
Proteins
His-His-His-His-His-His
Affinity Chromatography
Chromatography
Hantavirus Pulmonary Syndrome
Bunyaviridae
Isopropyl Thiogalactoside
Virus Assembly
Detergents
Sepharose
Nucleic Acids
Genome
RNA

All Science Journal Classification (ASJC) codes

  • Biotechnology

Cite this

Purification and characterization of the Sin Nombre virus nucleocapsid protein expressed in Escherichia coli. / Jonsson, Colleen; Gallegos, Juan; Fero, Phillip; Severson, William; Xu, Xiaolin; Schmaljohn, Connie S.

In: Protein Expression and Purification, Vol. 23, No. 1, 01.01.2001, p. 134-141.

Research output: Contribution to journalArticle

Jonsson, Colleen ; Gallegos, Juan ; Fero, Phillip ; Severson, William ; Xu, Xiaolin ; Schmaljohn, Connie S. / Purification and characterization of the Sin Nombre virus nucleocapsid protein expressed in Escherichia coli. In: Protein Expression and Purification. 2001 ; Vol. 23, No. 1. pp. 134-141.
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