RESUMO
Estrogen Receptor (ERalpha) is a member of superfamily of ligand-activated transcription factors which play critical roles in many biological processes. To screen novel modulators of ERalpha for drug development and biological function research, we developed a mammalian one-hybrid-based high-throughput screening model for ERalpha modulator. We cloned the ERalpha LBD gene from the total mRNA of fat tissue by RT-PCR and fused it with the GAL4 DNA binding domain of pBIND-GAL4 plasmid to construct a chimara expression plasmid pBIND-GAL4-Eralpha(LBD). The L02 cells was cotransfected with pBIND-GAL4-ERalpha(LBD) and a GAL4-responsive luciferase reporter plasmid pGL3-GAL4, and following treatment with test compounds for 24 h, the activities of luciferase were detected to evaluate the transactivities of ERalpha modulators. After manner optimizations of transfection conditions, Estradiol, an agonist control, induced the expression of luciferase in a dose-dependent with EC50 of 0.17 micromol/L, the maximum folds of induction was about 28.1. Tamoxifen, an antagonist control, efficiently suppressed the estradiol-mediated luciferase induction with EC50 of 0.10 micromol/L. Using this screening model, we discovered four ERalpha agonists from 2000 natural and synthetic compounds.
Assuntos
Animais , Humanos , Camundongos , Células 3T3-L1 , Quimera , Metabolismo , Proteínas de Ligação a DNA , Genética , Moduladores de Receptor Estrogênico , Química , Receptor alfa de Estrogênio , Genes Reporter , Genética , Genisteína , Química , Células HeLa , Luciferases , Genética , Metabolismo , Modelos Químicos , Proteínas de Saccharomyces cerevisiae , Genética , Fatores de Transcrição , Genética , TransfecçãoRESUMO
Glutathione S-transferase (GST) gene, PcgstA was cloned from the penicillin producing strain Penicillium chrysogenum,which is important for understanding the industrial fermentation process. PcgstA gene has an open-reading-frame of 840 bp in length,which is interrupted by two introns. The deduced amino acid sequence shows about 50% identity to several characterized filamentous fungi GSTs. The recombinant PcGSTA in Escherichia coli were overexpressed and purified. Enzymatic assays showed that the recombinant PcGSTA had a specific activity with 1-chloro-2, 4-dinitrobenzene of (0.159±0.031) μmol/(min· mg). It was found that the expression level of PcgstA in the penicillin producing medium supplemented with phenylacetic acid, the side chain precursor of penicillin G, was significant down regulated than that in medium without phenylacetic acid. This result suggested that PcGST may be related to phenylacetic acid metabolism in the penicillin producing strain.
