Expression and purification of recombinant Kaposi's sarcoma-associated herpesvirus DNA polymerase using a Baculovirus vector system.
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.
Laboratory of Antiviral Drug Mechanisms, SAIC-Frederick, P.O. Box B, Frederick, MD 21702, USA. dorj@ncifcrf.gov
Dorjbal
D
Dorjsuren
Yunden
Y
Badralmaa
Judy
J
Mikovits
Aiqun
A
Li
Robert
R
Fisher
Robert
R
Ricciardi
Robert
R
Shoemaker
Shizuko
S
Sei
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism*; D001671:Biochemistry / Q000379:methods; D002460:Cell Line; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000302:isolation & purification*; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005822:Genetic Vectors; D019288:Herpesvirus 8, Human / Q000378:metabolism*; D007313:Insecta; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification*
D003902:Detergents; D011994:Recombinant Proteins; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase