metallothionein and dermorphin

metallothionein has been researched along with dermorphin* in 1 studies

Other Studies

1 other study(ies) available for metallothionein and dermorphin

Article	Year
Expression of EGFP-amino-tagged human mu opioid receptor in Drosophila Schneider 2 cells: a potential expression system for large-scale production of G-protein coupled receptors. Protein expression and purification, 2003, Volume: 31, Issue:1 The G-protein coupled receptor (GPCR) human mu opioid receptor (hMOR) fused to the carboxy-terminus of the enhanced green fluorescent protein (EGFP) has been successfully and stably expressed in Drosophila Schneider 2 cells under the control of an inducible metallothionein promoter. Polyclonal cells expressing EGFPhMOR display high-affinity, saturable, and specific binding sites for the opioid antagonist diprenorphine. Competition studies with opioid agonists and antagonists defined the pharmacological profile of a mu opioid receptor similar to that observed in mammalian cells, suggesting proper folding of EGFPhMOR in a high-affinity state in Drosophila cells. The functionality of the fusion protein was demonstrated by the ability of agonist to reduce forskolin-stimulated cyclic AMP production and to induce [35S]GTPgammaS incorporation. The EGFPhMOR protein had the expected molecular weight (70kDa), as demonstrated by protein immunoblotting with anti-EGFP and anti-C-terminus hMOR antibodies. However, quantitative EGFP fluorescence intensity analysis revealed that the total level of expressed EGFPhMOR is 8-fold higher than the level of diprenorphine binding sites, indicating that part of the receptor is not in a high-affinity state. This may in part be due to a population of receptors localized in intracellular compartments, as shown by the distribution of fluorescence between the plasma membrane and the cell interior. This study shows that EGFP is a valuable and versatile tool for monitoring and quantifying expression levels as well as for optimizing and characterizing an expression system. Optimization of the Drosophila Schneider 2 cell expression system will allow large-scale purification of GPCRs, thus enabling structural studies to be undertaken. Topics: Animals; Binding, Competitive; Blotting, Western; Cell Line; Cloning, Molecular; Colforsin; Copper Sulfate; Cyclic AMP; Diprenorphine; DNA, Complementary; Drosophila; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Gene Expression; Genetic Vectors; Green Fluorescent Proteins; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Luminescent Proteins; Metallothionein; Microscopy, Confocal; Morphine; Naloxone; Naltrexone; Oligopeptides; Opioid Peptides; Pertussis Toxin; Polymerase Chain Reaction; Protein Binding; Receptors, G-Protein-Coupled; Receptors, Opioid, mu; Recombinant Fusion Proteins; Spectrometry, Fluorescence; Thermodynamics	2003

Article

Year

Expression of EGFP-amino-tagged human mu opioid receptor in Drosophila Schneider 2 cells: a potential expression system for large-scale production of G-protein coupled receptors.

Protein expression and purification, 2003, Volume: 31, Issue:1

The G-protein coupled receptor (GPCR) human mu opioid receptor (hMOR) fused to the carboxy-terminus of the enhanced green fluorescent protein (EGFP) has been successfully and stably expressed in Drosophila Schneider 2 cells under the control of an inducible metallothionein promoter. Polyclonal cells expressing EGFPhMOR display high-affinity, saturable, and specific binding sites for the opioid antagonist diprenorphine. Competition studies with opioid agonists and antagonists defined the pharmacological profile of a mu opioid receptor similar to that observed in mammalian cells, suggesting proper folding of EGFPhMOR in a high-affinity state in Drosophila cells. The functionality of the fusion protein was demonstrated by the ability of agonist to reduce forskolin-stimulated cyclic AMP production and to induce [35S]GTPgammaS incorporation. The EGFPhMOR protein had the expected molecular weight (70kDa), as demonstrated by protein immunoblotting with anti-EGFP and anti-C-terminus hMOR antibodies. However, quantitative EGFP fluorescence intensity analysis revealed that the total level of expressed EGFPhMOR is 8-fold higher than the level of diprenorphine binding sites, indicating that part of the receptor is not in a high-affinity state. This may in part be due to a population of receptors localized in intracellular compartments, as shown by the distribution of fluorescence between the plasma membrane and the cell interior. This study shows that EGFP is a valuable and versatile tool for monitoring and quantifying expression levels as well as for optimizing and characterizing an expression system. Optimization of the Drosophila Schneider 2 cell expression system will allow large-scale purification of GPCRs, thus enabling structural studies to be undertaken.

Topics: Animals; Binding, Competitive; Blotting, Western; Cell Line; Cloning, Molecular; Colforsin; Copper Sulfate; Cyclic AMP; Diprenorphine; DNA, Complementary; Drosophila; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Gene Expression; Genetic Vectors; Green Fluorescent Proteins; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Luminescent Proteins; Metallothionein; Microscopy, Confocal; Morphine; Naloxone; Naltrexone; Oligopeptides; Opioid Peptides; Pertussis Toxin; Polymerase Chain Reaction; Protein Binding; Receptors, G-Protein-Coupled; Receptors, Opioid, mu; Recombinant Fusion Proteins; Spectrometry, Fluorescence; Thermodynamics

2003