title: Expression, purification, and enzymatic characterization of Bombyx mori nucleopolyhedrovirus DNA polymerase.
abstract: Bombyx mori nucleopolyhedrovirus (BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms. BmNPV DNA polymerase (Bm-DNAPOL), encoded by the ORF53 gene, plays a central role in viral DNA replication. In this work, a His-tagged Bm-DNAPOL fusion protein, constructed using a novel MultiBac expression system, was overexpressed in Sf-9 insect cells, purified to near homogeneity on Ni-NTA agarose beads and further purified by ion-exchange chromatography. About 0.4 mg of enzyme was obtained from about 1 × 10(9) infected Sf-9 cells in suspension culture. Characterization of the highly purified enzyme indicated that Bm-DNAPOL is a monomer with an apparent molecular mass of approximately 110,000 Da. It possessed a specific activity of 15,126.3 U/mg under optimal in vitro reaction conditions and behaved in the manner of a proliferating cell nuclear antigen (PCNA)-independent DNA polymerase on both poly(dA)/oligo(dT) primer/template and singly premiered M13 DNA. BmNPV viral replication may be independent of replication factor C and a PCNA complex, while single-stranded DNA binding protein might play an important role in BmNPV DNA replication. These findings will be significant in studies on BmNPV-based disease in silkworms and for using silkworms as a bioreactor for the production of biomolecules of commercial importance.
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang, 212013, Jiangsu, People's Republic of China.
authors.forename: Liu
authors.initials: L
authors.lastname: Liu
authors.forename: Huifang
authors.initials: H
authors.lastname: Song
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Xiaoting
authors.initials: X
authors.lastname: Fan
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhang
authors.forename: Keping
authors.initials: K
authors.lastname: Chen
authors.forename: Huiqing
authors.initials: H
authors.lastname: Chen
authors.forename: Yajing
authors.initials: Y
authors.lastname: Zhou
mesh_terms: D000818:Animals; D012831:Bombyx / Q000821:virology; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015870:Gene Expression; D008970:Molecular Weight; D017924:Nucleopolyhedroviruses / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D061987:Sf9 Cells
chemical_list: D011993:Recombinant Fusion Proteins; D004259:DNA-Directed DNA Polymerase