U.S. Department of Defense


Date of this Version



Protein Expression and Purification 23, 134-141 (2001) doi:1O,1006/prep,2001.1489


This article is a U.S. government work, and is not subject to copyright in the United States.


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 IPTC (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 Az80 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. Hantaviruses cause two illnesses in humans, hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) (1). Hantaan virus (HTNV) and Sin Nombre virus (SNV) are the major causative agents of HFRS and HCPS, respectively. Hantaviruses have a negative-sense, singlestranded RNA genome that consists of three segments, S, M, and L (2, 3). Transcription of each vRNA by the viral-encoded RNA-dependent RNA polymerase (RdRp) yields a complementary RNA (cRNA), which in turn is used as a template to generate vRNA by the RdRp. In contrast to the hantaviral mRNAs, which do not associate with the viral nucleocapsid protein (N) (4), the vRNA and cRNA replicative templates are encapsidated by N. The interactions of the hantaviral RNAs and N are not well defined. Gott et a1. showed the presence of a nonspecific RNA binding domain in the Cterminus of the HTNV N protein (5). In another study, the HTNV N bound preferentially to the viral genome rather than to nonviral RNA (6). More extensive biochemical studies of RNA-protein or protein-protein interactions will require sufficient quantities of highly purified, soluble, monodispersed N protein that are free of contaminating ribonucleases, nucleic acids, and RNA binding proteins. Toward this goal, we have investigated methods to recover hantaviral N from a bacterial expression system in a form suitable for biochemical analysis. Our previous purification strategy, as well as that of others, relied on using denaturing methods to recover soluble expressed N from bacteria (5, 6). Because renaturing does not always result in native protein, large losses in functional N are common. Comparing yields of HTNV and SNV N after denaturing and renaturing shows that the SNV N protein is more readily recovered and probably is more stable (6). No obvious reason for the difference in ease of recovery of soluble SNV N versus HTNV N can be deduced from examining the amino acid sequences of the two proteins. The most notable difference is the substitution of seven Gly residues in the HTNV N protein with Asn, Ala, Ser, or Asp in the SNV N protein. All of these substituted amino acids are more hydrophilic than Gly (7), and if they are located on the surface of the protein, they could facilitate the interaction ofthe protein with an aqueous environment. Insight into the molecular basis for the differences awaits a three-dimensional structure that could provide information on the solvent accessibility of these residues. Herein, we report studies leading to the development of a rapid soluble extraction protocol and chromatography method for recovery and purification of the SNV N protein expressed in Escherichia coli. We further test this method for suitability for preparing N proteins of three other hantaviruses