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

  1. Author:
    Dorjsuren, D.
    Badralmaa, Y.
    Mikovits, J.
    Li, A. Q.
    Fisher, R.
    Ricciardi, R.
    Shoemaker, R.
    Sei, S.

  2. Author Address

    SAIC Frederick, Lab Antiviral Drug Mech, POB B, Ft Detrick, MD 21702 USA SAIC Frederick, Lab Antiviral Drug Mech, Ft Detrick, MD 21702 USA SAIC Frederick, Clin Serv Program, Lab Mol Cell Biol, Ft Detrick, MD 21702 USA SAIC Frederick, Prot Chem Lab, Ft Detrick, MD 21702 USA Univ Penn, Dept Microbiol, Philadelphia, PA USA Univ Penn, Dept Biochem & Biophys, Philadelphia, PA USA NCI Frederick, Screening Technol Branch, Dev Therapeut Program, Ft Detrick, MD 21702 USA Dorjsuren D SAIC Frederick, Lab Antiviral Drug Mech, POB B, Ft Detrick, MD 21702 USA
    1. Year: 2003
  2. Journal: Protein Expression and Purification
    1. 29
    2. 1
    3. Pages: 42-50
  4. Type of Article: Article
  1. 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 antiherpetic 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 FITS of large chemical libraries to identify novel KSHV DNA polymerase inhibitors. (C) 2003 Elsevier Science (USA). All rights reserved.

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