Budded baculovirus particles as a source of membrane proteins for radioligand binding assay: The case of dopamine D 1 receptor
| Autor: | Anni Allikalt, Ago Rinken |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Pharmacology Chemistry Ligand binding assay Sf9 Toxicology 03 medical and health sciences 030104 developmental biology 0302 clinical medicine Membrane Biochemistry Membrane protein Cell surface receptor Radioligand Receptor 030217 neurology & neurosurgery G protein-coupled receptor |
| Zdroj: | Journal of Pharmacological and Toxicological Methods. 86:81-86 |
| ISSN: | 1056-8719 |
| DOI: | 10.1016/j.vascn.2017.04.004 |
| Popis: | Introduction G-protein-coupled receptors have become very important drug targets and therefore ligand binding assays for these receptors are an essential part of drug discovery. Among a variety of experimental systems, the radioligand binding assay has remained as one of the main methods in this field for decades. Usually cell membranes or tissues are used in these experiments, however in this article we demonstrate that baculoviruses produced in Sf9 cells display recombinant receptors on their surface and therefore can be used in radioligand binding assay. Methods We have used baculoviruses with dopamine D 1 receptors as a model system to validate the usage of this receptor preparation in radioligand binding experiments. In order to collect membrane receptors and separate free radioligand, we have applied FilterMate Harvester with Filtermat B. Results Using baculoviruses with dopamine D 1 receptors as a model system, we have shown that this is a suitable preparation for conventional radioligand binding assay. Here, all the experiments were performed using the dopamine D 1 -like receptor specific radioligand [ 3 H]SCH23390. There were no significant differences between binding parameters determined in membranes of Sf9 cells and baculovirus particles. Similar pIC 50 and K i values were also determined in competition binding assays with HEK293 cell membranes. Discussion All the results obtained with baculovirus preparation were in good agreement with the data obtained in parallel experiments with membrane preparations from Sf9 and HEK293 cells expressing dopamine D 1 receptors. Shape uniformity, homogeneous distribution and slow sedimentation of the membranes are some of the advantages of baculovirus preparations, which prove them as promising source of membrane proteins for routine and high throughput analysis. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect