TY - JOUR
T1 - Purification and characterization of the major nonstructural protein (NS-1) of Aleutian mink disease parvovirus.
AU - Christensen, Jesper Aagaard
AU - Pedersen, M
AU - Aasted, B
AU - Alexandersen, S
N1 - Keywords: Adenosine Triphosphatases; Aleutian Mink Disease Virus; Animals; Antibodies, Monoclonal; Antigens, Viral; Cations, Divalent; Cloning, Molecular; DNA Helicases; Genes, Viral; Moths; Mutagenesis, Site-Directed; Nucleopolyhedrovirus; Recombinant Proteins; Solubility; Structure-Activity Relationship; Viral Nonstructural Proteins; Viral Structural Proteins
PY - 1995
Y1 - 1995
N2 - We have previously described the expression of the major nonstructural protein (NS-1) of Aleutian mink disease parvovirus (ADV) in insect cells by using a baculovirus vector (J. Christensen, T. Storgaard, B. Bloch, S. Alexandersen, and B. Aasted, J. Virol. 67:229-238, 1993). To study its biochemical properties, ADV NS-1 was expressed in Sf9 insect cells and purified to apparent homogeneity with a combination of nuclear extraction, Zn2+ ion chromatography, and immunoaffinity chromatography on monoclonal antibodies. The purified protein showed ATP binding and ATPase- and ATP- or dATP-dependent helicase activity requiring either Mg2+ or Mn2+ as a cofactor. The ATPase activity of NS-1 was efficiently stimulated by single-stranded DNA and, to a lesser extent, double-stranded DNA. We also describe the expression, purification, and characterization of a mutant NS-1 protein, in which a lysine in the putative nucleotide binding consensus sequence of the molecule was replaced with serine. The mutated NS-1 was expressed at 10-fold higher levels than wild-type NS-1, but it exhibited no ATP binding. ATPase, or helicase activity. The availability of large amounts of purified functional NS-1 protein will facilitate studies of the biochemistry of ADV replication and gene regulation leading to disease in mink.
AB - We have previously described the expression of the major nonstructural protein (NS-1) of Aleutian mink disease parvovirus (ADV) in insect cells by using a baculovirus vector (J. Christensen, T. Storgaard, B. Bloch, S. Alexandersen, and B. Aasted, J. Virol. 67:229-238, 1993). To study its biochemical properties, ADV NS-1 was expressed in Sf9 insect cells and purified to apparent homogeneity with a combination of nuclear extraction, Zn2+ ion chromatography, and immunoaffinity chromatography on monoclonal antibodies. The purified protein showed ATP binding and ATPase- and ATP- or dATP-dependent helicase activity requiring either Mg2+ or Mn2+ as a cofactor. The ATPase activity of NS-1 was efficiently stimulated by single-stranded DNA and, to a lesser extent, double-stranded DNA. We also describe the expression, purification, and characterization of a mutant NS-1 protein, in which a lysine in the putative nucleotide binding consensus sequence of the molecule was replaced with serine. The mutated NS-1 was expressed at 10-fold higher levels than wild-type NS-1, but it exhibited no ATP binding. ATPase, or helicase activity. The availability of large amounts of purified functional NS-1 protein will facilitate studies of the biochemistry of ADV replication and gene regulation leading to disease in mink.
M3 - Journal article
C2 - 7853520
SN - 0022-538X
VL - 69
SP - 1802
EP - 1809
JO - Journal of Virology
JF - Journal of Virology
IS - 3
ER -