Purification and characterization of recombinant human respiratory syncytial virus nonstructural protein NS1.
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.
Ling
Zhenhua
Z
Graduate Program in Biochemistry, Microbiology, and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.
Tran
Kim C
KC
Arnold
Jamie J
JJ
Teng
Michael N
MN
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*
C471213:His-His-His-His-His-His; D007106:Immune Sera; D009842:Oligopeptides; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D006639:Histidine