Expression and purification of recombinant Kaposi's sarcoma-associated herpesvirus DNA polymerase using a Baculovirus vector system

Protein Expr Purif. 2003 May;29(1):42-50. doi: 10.1016/s1046-5928(03)00017-2.

Abstract

The DNA polymerase (POL) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for viral DNA replication and, thus, may be considered as a viable target for anti-KSHV therapeutics. To produce large quantities of homogeneous and pure POL required for high-throughput screening (HTS) for inhibitors, we generated a recombinant baculovirus vector encoding a hexahistidine (His6)-tagged POL and infected Spodoptera frugiperda Sf-9 insect cells. High expression of recombinant POL (rPOL) was achieved for up to 72h post-infection. The rPOL was solubilized in lysis buffer containing 0.3% Cymal-5 detergent, purified by metal-chelating and dsDNA-cellulose affinity chromatography, and analyzed by anti-His antibody Western blot and mass spectrometry. The functionality of rPOL was confirmed by its DNA synthesis activity in vitro, which was effectively blocked by the anti-herpetic DNA polymerase inhibitors, foscarnet and cidofovir diphosphate, in a dose-dependent manner. The POL expressed and purified from the recombinant baculovirus-infected insect cells may be useful toward the development of HTS of large chemical libraries to identify novel KSHV DNA polymerase inhibitors.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Baculoviridae / metabolism*
  • Biochemistry / methods
  • Cell Line
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / isolation & purification*
  • Detergents / pharmacology
  • Dose-Response Relationship, Drug
  • Genetic Vectors
  • Herpesvirus 8, Human / metabolism*
  • Insecta
  • Mass Spectrometry
  • Molecular Sequence Data
  • Recombinant Proteins / chemistry
  • Sequence Homology, Amino Acid
  • Viral Proteins / chemistry
  • Viral Proteins / isolation & purification*

Substances

  • Detergents
  • Recombinant Proteins
  • Viral Proteins
  • DNA-Directed DNA Polymerase