Expression of Minute Virus of Mice Major Nonstructural Protein in Insect Cells: Purification and Identification of ATPase and Helicase Activities

Virology. 1991 Nov;185(1):90-8. doi: 10.1016/0042-6822(91)90757-3.

Abstract

The gene encoding the major nonstructural (NS-1) protein of minute virus of mice (MVM) has been expressed in insect cells using a baculovirus expression system. This 83-kDa polypeptide was found to be localized in the soluble (cytosolic) fraction in insect cells, in contrast with the nuclear localization of NS-1 expressed in MVM-infected mouse LA-9 cells. The protein was purified by immunoaffinity chromatography using a monoclonal antibody (MAb) prepared to an NS-1 fusion peptide [(Yeung et al., Virology 185, 35-45 (1991)]. Recombinant NS-1 was eluted using either low pH or a synthetic peptide corresponding to the epitope of the MAb. The peptide-eluted material is greater than 95% pure and biologically active in that it has ATPase activity and ATP-dependent helicase activity as determined by a strand displacement assay.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / genetics*
  • Adenosine Triphosphatases / isolation & purification
  • Adenosine Triphosphatases / metabolism
  • Adenosine Triphosphate / metabolism
  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal
  • Baculoviridae / genetics
  • Capsid / genetics*
  • Capsid / isolation & purification
  • Capsid / metabolism
  • Cell Line
  • Chromatography, Affinity
  • Cloning, Molecular
  • Deoxyadenine Nucleotides / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Genes, Viral
  • Insecta
  • Minute Virus of Mice / enzymology
  • Minute Virus of Mice / genetics*
  • Molecular Sequence Data
  • Molecular Weight
  • Multienzyme Complexes / genetics
  • Multienzyme Complexes / isolation & purification
  • Multienzyme Complexes / metabolism
  • Peptides / chemical synthesis
  • Peptides / immunology
  • Plasmids
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • Transfection*
  • Viral Core Proteins / genetics*
  • Viral Core Proteins / isolation & purification
  • Viral Core Proteins / metabolism
  • Viral Nonstructural Proteins

Substances

  • Antibodies, Monoclonal
  • Deoxyadenine Nucleotides
  • Multienzyme Complexes
  • Peptides
  • Recombinant Proteins
  • Viral Core Proteins
  • Viral Nonstructural Proteins
  • Adenosine Triphosphate
  • Adenosine Triphosphatases
  • 2'-deoxyadenosine triphosphate