title: Purification and characterization of recombinant human respiratory syncytial virus nonstructural protein NS1.
abstract: We report here the first biochemical and structural characterization of the respiratory syncytial virus (RSV) NS1 protein. We have used a pET-ubiquitin expression system to produce respiratory syncytial virus (RSV) NS1 protein in E. coli that contains a hexahistidine-tag on either the amino- or carboxyl-terminus (His(6)-NS1 and NS1-His(6), respectively). We have been able to isolate milligram quantities of highly purified His(6)-NS1 and NS1-His(6) by nickel affinity chromatography. Generation of recombinant RSV indicated that addition of the hexahistidine tag to the C-terminus of NS1 slightly decreased viral replication competence whereas addition of the tag to the N-terminus had no observable effect. Therefore, we performed a comprehensive biochemical and biophysical characterization on His(6)-NS1. His(6)-NS1 is monodisperse in solution as determined by dynamic light scattering analysis. Both gel filtration and analytical ultracentrifugation showed that His(6)-NS1 is predominantly a monomer. In agreement with theoretical predictions, circular dichroism spectroscopy showed that His(6)-NS1 contains 21% alpha-helices, 34% beta-sheets, and 45% undefined structure. Immunization with purified His(6)-NS1 generated an antiserum that specifically recognizes NS1 by immunoprecipitation from HEp-2 cells infected by RSV, indicating that His(6)-NS1 resembles native NS1. The availability of purified RSV NS1 will permit biochemical and structural investigations providing insight into the function of NS1 in viral replication and interferon antagonism.
authors.affiliation: Graduate Program in Biochemistry, Microbiology, and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Zhenhua
authors.initials: Z
authors.lastname: Ling
authors.forename: Kim C
authors.initials: KC
authors.lastname: Tran
authors.forename: Jamie J
authors.initials: JJ
authors.lastname: Arnold
authors.forename: Michael N
authors.initials: MN
authors.lastname: Teng
mesh_terms: D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004926:Escherichia coli; D006639:Histidine / Q000378:metabolism; D006801:Humans; D007106:Immune Sera; D008027:Light; D009842:Oligopeptides / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D018113:Respiratory Syncytial Virus, Human / Q000737:chemistry*; D012542:Scattering, Radiation; D014461:Ultracentrifugation; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: C471213:His-His-His-His-His-His; D007106:Immune Sera; D009842:Oligopeptides; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D006639:Histidine