title: Characterization and purification of human retinoic acid receptor-gamma 1 overexpressed in the baculovirus-insect cell system.
abstract: The full-length cDNA for the human retinoic acid receptor-gamma 1 (RAR-gamma 1) has been expressed to high levels in Spodoptera frugiferda (Sf9) cells using the baculovirus expression system. Western blot analysis revealed that RAR-gamma 1 expression increased between 32 and 60 h post-infection. The recombinant receptor was expressed primarily as a nuclear protein and displayed a molecular mass of 50 kDa as determined by SDS/PAGE and gel-filtration chromatography, consistent with its cDNA-deduced size. Based on ligand binding, 2 x 10(6) RAR-gamma 1 molecules were expressed per Sf9 cell, a level approx. 2000 times greater than in mammalian cells. The receptor was partially purified 300-fold by sequential anion-exchange, gel-filtration and DNA affinity chromatographies. The overexpressed receptor specifically bound all-trans-retinoic acid (RA) and the synthetic retinoid CD367 with high affinity (Kd 0.15 nM and 0.23 nM respectively). The RA metabolites 4-hydroxy-RA and 4-oxo-RA were poor competitors for [3H]CD367 binding to recombinant RAR-gamma 1 (K(i) > 1 microM), indicating that 4-oxidation of RA greatly reduces its affinity for RAR-gamma 1. Gel-retardation analysis demonstrated that RAR-gamma 1 specifically bound the RA response element of the mouse RAR-beta gene. RAR-gamma 1 species expressed from recombinant baculovirus (in Sf9 cells) and vaccinia virus (in HeLa cells) exhibited similar affinities for RA and CD367 and had comparable DNA-binding properties in gel-retardation experiments. Moreover, a similar requirement for additional DNA-binding stimulatory factor(s) was observed in both cases. These results provide a basis for the use of baculovirus-expressed RAR-gamma 1 in further functional and structural studies.
authors.affiliation: Department of Dermatology, University of Michigan Medical School, Ann Arbor 48109.
authors.forename: A P
authors.initials: AP
authors.lastname: Reddy
authors.forename: J Y
authors.initials: JY
authors.lastname: Chen
authors.forename: T
authors.initials: T
authors.lastname: Zacharewski
authors.forename: H
authors.initials: H
authors.lastname: Gronemeyer
authors.forename: J J
authors.initials: JJ
authors.lastname: Voorhees
authors.forename: G J
authors.initials: GJ
authors.lastname: Fisher
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002645:Chickens; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009036:Moths; D011485:Protein Binding; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014212:Tretinoin / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D008024:Ligands; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D014212:Tretinoin; D004247:DNA