Optimizing the Expression of Human Dopamine Receptors in Escherichia coli.
The human dopamine receptors D2S and D3 belong to the group of G protein-coupled receptors (GPCRs) and are important drug targets. Structural analyses and development of new receptor subtype specific drugs have been impeded by low expression yields or receptor instability. Fusing the T4 lysozyme into the intracellular loop 3 improves crystallization but complicates conformational studies. To circumvent these problems, we expressed the human D2S and D3 receptors in Escherichia coli using different N- and C-terminal fusion proteins and thermostabilizing mutations. We optimized expression times and used radioligand binding assays with whole cells and membrane homogenates to evaluate KD-values and the number of receptors in the cell membrane. We show that the presence but not the type of a C-terminal fusion protein is important. Bacteria expressing receptors capable of ligand binding can be selected using FACS analysis and a fluorescently labeled ligand. Improved receptor variants can thus be generated using error-prone PCR. Subsequent analysis of clones showed the distribution of mutations over the whole gene. Repeated cycles of PCR and FACS can be applied for selecting highly expressing receptor variants with high affinity ligand binding, which in the future can be used for analytical studies.
['Boritzki|Vanessa|V|', 'Hübner|Harald|H|', 'Allikalt|Anni|A|', 'Gmeiner|Peter|P|0000-0002-4127-197X', 'Wöhrl|Birgitta M|BM|0000-0002-6024-9487']
D002138:Calibration D002462:Cell Membrane D003001:Cloning, Molecular D004926:Escherichia coli D015723:Gene Library D006801:Humans D009154:Mutation D030781:Organisms, Genetically Modified D015202:Protein Engineering D011954:Receptors, Dopamine D017448:Receptors, Dopamine D2 D050637:Receptors, Dopamine D3 D043562:Receptors, G-Protein-Coupled D011994:Recombinant Proteins D014169:Transformation, Bacterial D019076:Transgenes
human dopamine receptors GPCR radioligand binding FACS expression in E. coli fluorescent ligand gene library protein engineering