Purification and characterization of recombinant human respiratory syncytial virus nonstructural protein NS1

Zhenhua Ling, Kim C. Tran, Jamie J. Arnold, Michael N. Teng

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

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 (His6-NS1 and NS1-His6, respectively). We have been able to isolate milligram quantities of highly purified His6-NS1 and NS1-His6 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 His6-NS1. His6-NS1 is monodisperse in solution as determined by dynamic light scattering analysis. Both gel filtration and analytical ultracentrifugation showed that His6-NS1 is predominantly a monomer. In agreement with theoretical predictions, circular dichroism spectroscopy showed that His6-NS1 contains 21% α-helices, 34% β-sheets, and 45% undefined structure. Immunization with purified His6-NS1 generated an antiserum that specifically recognizes NS1 by immunoprecipitation from HEp-2 cells infected by RSV, indicating that His6-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.

Original languageEnglish (US)
Pages (from-to)261-270
Number of pages10
JournalProtein Expression and Purification
Volume57
Issue number2
DOIs
StatePublished - Feb 1 2008

All Science Journal Classification (ASJC) codes

  • Biotechnology

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