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Expression, purification, and enzymatic characterization of Bombyx mori nucleopolyhedrovirus DNA polymerase.
- Source :
-
Archives of Virology . Dec2013, Vol. 158 Issue 12, p2453-2463. 11p. - Publication Year :
- 2013
-
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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 03048608
- Volume :
- 158
- Issue :
- 12
- Database :
- Academic Search Index
- Journal :
- Archives of Virology
- Publication Type :
- Academic Journal
- Accession number :
- 91996718
- Full Text :
- https://doi.org/10.1007/s00705-013-1758-8