Expression, purification, and enzymatic characterization of Bombyx mori nucleopolyhedrovirus DNA polymerase

Expression, purification, and enzymatic characterization of Bombyx mori nucleopolyhedrovirus DNA... Bombyx mori nucleopolyhedrovirus ( Bm NPV) is a major viral agent that causes deadly grasserie disease in silkworms. Bm NPV 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. Bm NPV 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 Bm NPV DNA replication. These findings will be significant in studies on Bm NPV-based disease in silkworms and for using silkworms as a bioreactor for the production of biomolecules of commercial importance. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Virology Springer Journals

Expression, purification, and enzymatic characterization of Bombyx mori nucleopolyhedrovirus DNA polymerase

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Publisher
Springer Journals
Copyright
Copyright © 2013 by Springer-Verlag Wien
Subject
Biomedicine; Virology; Medical Microbiology; Infectious Diseases
ISSN
0304-8608
eISSN
1432-8798
D.O.I.
10.1007/s00705-013-1758-8
Publisher site
See Article on Publisher Site

Abstract

Bombyx mori nucleopolyhedrovirus ( Bm NPV) is a major viral agent that causes deadly grasserie disease in silkworms. Bm NPV 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. Bm NPV 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 Bm NPV DNA replication. These findings will be significant in studies on Bm NPV-based disease in silkworms and for using silkworms as a bioreactor for the production of biomolecules of commercial importance.

Journal

Archives of VirologySpringer Journals

Published: Dec 1, 2013

References

  • Molecular phylogeny of silkmoths reveals the origin of domesticated silkmoth, Bombyx mori from Chinese Bombyx mandarina and paternal inheritance of Antheraea proylei mitochondrial DNA
    Arunkumar, KP; Metta, M; Nagaraju, J
  • Gene expression profiling of resistant and susceptible Bombyx mori strains reveals nucleopolyhedrovirus-associated variations in host gene transcript levels
    Bao, YY; Tang, XD; Lv, ZY; Wang, XY; Tian, CH; Xu, YP; Zhang, CX

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