Expression of minute virus of mice major nonstructural protein in insect cells: purification and identification of ATPase and helicase activities.
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.
Wilson
G M
GM
Department of Biochemistry, Faculty of Medicine, University of British Columbia, Vancouver, Canada.
Jindal
H K
HK
Yeung
D E
DE
Chen
W
W
Astell
C R
CR
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae / Q000235:genetics; D002213:Capsid / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005814:Genes, Viral; D007313:Insecta; D008915:Minute Virus of Mice / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D014758:Viral Core Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017361:Viral Nonstructural Proteins
D000911:Antibodies, Monoclonal; D003838:Deoxyadenine Nucleotides; D009097:Multienzyme Complexes; D010455:Peptides; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C026600:2'-deoxyadenosine triphosphate