scholarly journals Characterization and purification of human retinoic acid receptor-γ 1 overexpressed in the baculovirus-insect cell system

1992 ◽  
Vol 287 (3) ◽  
pp. 833-840 ◽  
Author(s):  
A P Reddy ◽  
J Y Chen ◽  
T Zacharewski ◽  
H Gronemeyer ◽  
J J Voorhees ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Retinoic Acid Receptor ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Sf9 Cells ◽  
Acid Receptor ◽  
Gel Retardation

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.

Biochemical Chgaracterization of Recombinant Baculovirus 373 K/E p53 Protein

2018 ◽  
Vol 9 (03) ◽  
pp. 20204-20223
Author(s):  
Maghsoudi, Hossein ◽  
U Pati
Keyword(s):  
Dna Binding ◽  
P53 Protein ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Cross Linking ◽  
Mobility Shift ◽  
Dna Complexes ◽  
P53 Antibodies

In this study, we expressed and purified the recombinant baculovirus 373 K/E p53 protein in a baculovirus expression system to characterize this mutant and compare it with wild type p53. Gel- filtration chromatography and chemical cross-linking experiments indicated that purified recombinant baculovirus 373 K/E p53 protein assembles into multimeric forms ranging from tetramers to polymers. Gel-mobility shift assays and protein-DNA cross-linking studies demonstrated that the recombinant protein binds, to a consensus DNA target as a dimer but that additional p53 mutant molecules may then associate with the preformed p53-dimer-DNA complexes to form a larger p53_DNA complexes. These observations suggest that the p53 mutant tetramers and polymers that forms the minimal p53 mutant complex in solution dissociated upon DNA binding to form p53 mutant dimmer DNA complexes. The DNA binding activity of this mutant was then investigated using electrophoretic mobility shift assays as well as supershift assay with anti-p53 antibodies. Binding of the anti-p53 antibody PAb421to the oligomerization promoting domain on p53 stimulated the sequential formation of both the p53_dimer DNA and larger p53-DNA complexes

Production and characterization of a retinoic acid receptor RARγ construction encompassing the DNA binding domain and the disordered N-terminal proline rich domain

2014 ◽  
Vol 95 ◽  
pp. 113-120 ◽  
Author(s):  
Denise Martinez-Zapien ◽  
Marc-André Delsuc ◽  
Gilles Travé ◽  
Régis Lutzing ◽  
Cécile Rochette-Egly ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Dna Binding ◽  
Retinoic Acid Receptor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Acid Receptor ◽  
Rich Domain

scholarly journals Ligand-Activated Retinoic Acid Receptor Inhibits AP-1 Transactivation by Disrupting c-Jun/c-Fos Dimerization

1999 ◽  
Vol 13 (2) ◽  
pp. 276-285 ◽  
Author(s):  
Xiao-Feng Zhou ◽  
Xi-Qiang Shen ◽  
Lirim Shemshedini
Keyword(s):  
Retinoic Acid ◽  
Dna Binding ◽  
Transcriptional Activity ◽  
Retinoic Acid Receptor ◽  
Cellular Growth ◽  
Dependent Manner ◽  
Acid Receptor ◽  
Retinoid Receptors ◽  
Two Hybrid

Abstract In the presence of retinoic acid (RA), the retinoid receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), are able to up-regulate transcription directly by binding to RA-responsive elements on the promoters of responsive genes. Liganded RARs and RXRs are also capable of down-regulating transcription, but, by contrast, this is an indirect effect, mediated by the interaction of these nuclear receptors not with DNA but the transcription factor activating protein-1 (AP-1). AP-1 is a dimeric complex of the protooncoproteins c-Jun and c-Fos and directly regulates transcription of genes important for cellular growth. Previous in vitro results have suggested that RARs can block AP-1 DNA binding. Using a mammalian two-hybrid system, we report here that human RARα (hRARα) can disrupt in a RA-dependent manner the homo- and heterodimerization properties of c-Jun and c-Fos. This inhibition of dimerization is cell specific, occurring only in those cells that exhibit RA-induced repression of AP-1 transcriptional activity. Furthermore, this mechanism appears to be specific for the RARs, since another potent inhibitor of AP-1 activity, the glucocorticoid receptor, does not affect AP-1 dimerization. Our data argue for a novel mechanism by which RARs can repress AP-1 DNA binding, in which liganded RARs are able to interfere with c-Jun/c-Jun homodimerization and c-Jun/c-Fos heterodimerization and, in this way, may prevent the formation of AP-1 complexes capable of DNA binding.

scholarly journals Identification of Amino Acids Critical for the DNA Binding and Dimerization Properties of the Human Retinoic Acid Receptor α

1996 ◽  
Vol 271 (30) ◽  
pp. 17996-18006 ◽  
Author(s):  
Christophe Rachez ◽  
Pierre Sautière ◽  
Pierre Formstecher ◽  
Philippe Lefebvre
Keyword(s):  
Amino Acids ◽  
Retinoic Acid ◽  
Dna Binding ◽  
Retinoic Acid Receptor ◽  
Acid Receptor ◽  
Retinoic Acid Receptor Α

The DNA binding domain of retinoic acid receptor beta is required for ligand-dependent suppression of proliferation. Application of general purpose mammalian coexpression vectors

1994 ◽  
Vol 107 (4) ◽  
pp. 827-838 ◽  
Author(s):  
J.V. Frangioni ◽  
N. Moghal ◽  
A. Stuart-Tilley ◽  
B.G. Neel ◽  
S.L. Alper
Keyword(s):  
Cell Proliferation ◽  
Retinoic Acid ◽  
Dna Binding ◽  
Retinoic Acid Receptor ◽  
General Purpose ◽  
Acid Receptor ◽  
All Trans Retinoic Acid ◽  
Retinoic Acid Receptor Beta ◽  
Transient Overexpression ◽  
Receptor Beta

We have developed a family of mammalian coexpression vectors that permit identification of living or fixed cells overexpressing a gene of interest by surrogate detection of a coexpressed marker protein. Using these ‘pMARK’ vectors, a fluorescence-based, single cell proliferation assay was developed and used to study the effect of retinoic acid receptor beta (RAR-beta) on cell cycling. We demonstrate that transient overexpression of RAR-beta in the presence, but not absence, of all-trans retinoic acid results in a dramatic suppression of cell proliferation. We further show that this effect requires the DNA binding (C) domain of RAR-beta. It has been previously shown that RAR-beta expression is markedly altered in a variety of neoplasms and cell lines. Our data support the hypothesis that loss of RAR-beta may contribute to tumor progression by removing normal restraints on proliferation. The pMARK vectors should be useful for studying other genes that putatively suppress or enhance proliferation.

scholarly journals Ligation of Retinoic Acid Receptor α Regulates Negative Selection of Thymocytes by Inhibiting Both DNA Binding of nur77 and Synthesis of Bim

2003 ◽  
Vol 170 (7) ◽  
pp. 3577-3584 ◽  
Author(s):  
Éva Szegezdi ◽  
Ildikó Kiss ◽  
Ágnes Simon ◽  
Bernadett Blaskó ◽  
Uwe Reichert ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Dna Binding ◽  
Negative Selection ◽  
Retinoic Acid Receptor ◽  
Acid Receptor ◽  
Retinoic Acid Receptor Α ◽  
Selection Of
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