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The screening of ligands for membrane receptor proteins is central to the discovery of new pharmaceutical drugs. We present a general method to reversibly attach receptor proteins via an affinity tag to a quartz surface and subsequently detect with high sensitivity the real-time binding of ligands by total internal reflection fluorescence. A serotonin-gated(More)
Understanding cellular signaling mediated by cell surface receptors is key to modern biomedical research and drug development. The discovery of a growing number of potential molecular targets and therapeutic compounds requires downscaling and accelerated functional screening. Receptor-mediated cellular responses are typically investigated on single cells or(More)
The expression of recombinant proteins with the Semliki Forest Virus (SFV) system has been scaled up to bioreactor scale. As a model protein for this study the human 5-HT(3) receptor was chosen. The gene for the receptor was subcloned into the SFV expression plasmid pSFV1. Virus production by in vivo packaging and production of the recombinant protein was(More)
Fluorescent 2'-deoxynucleotides containing a protecting group at the 3'-O-position are reversible terminators enabling array-based DNA sequencing by synthesis (SBS) approaches. Herein, we describe the synthesis of a new family of 3'-OH unprotected cleavable fluorescent 2'-deoxynucleotides and their evaluation as reversible terminators for high-throughput(More)
A serotonin 5-HT3 receptor was functionally expressed to high levels and on a large scale in mammalian cells with the Semliki Forest virus system. Conditions were optimized to maximize detergent solubilization of the receptor, while preserving ligand binding activity. An efficient one-step purification yielding approximately 50% of the histidine-tagged(More)
We have measured fluorescence resonance energy transfer (FRET) between a fluorescent antagonist, bound to the purified detergent-solubilized serotonin type 3 receptor, and a lipophilic acceptor probe partitioned into the micelle surrounding the detergent-solubilized receptor. The experimentally observed FRET efficiency was evaluated on the basis of the(More)
In this paper, an Ant Colony Optimization Algorithm (ACO) is proposed for operations of steady flow gas pipeline. The system is composed of compressing stations linked by pipe legs. The decisions variables are chosen to be the operating turbo compressor number and the discharge pressure for each compressing station. The objective function is the power(More)
A fluorescently labelled ligand for the 5-HT3 serotonin receptor was synthesised and its sub-nanomolar affinity for the purified, detergent solubilised receptor was measured. The change in the ligand's fluorescence upon receptor binding was used to directly measure its dissociation constant for receptor binding, to determine the pharmacology of the(More)
The thermodynamics and kinetics of ligand binding to the purified serotonin 5HT3 receptor and the local environment of the bound ligand were studied by fluorescence spectroscopy using a novel fluorescein-labeled ligand GR-flu [1,2,3, 9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-amino-(N-fluo rescien-thiocarbamoyl)-propyl)-4H-carbazol-4-one].(More)