Optimizing the Expression of Human Dopamine Receptors in <i>Escherichia coli</i>
The human dopamine receptors D<sub>2S</sub> and D<sub>3</sub> 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 re...
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MDPI AG
2021-08-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/16/8647 |
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| author | Vanessa Boritzki Harald Hübner Anni Allikalt Peter Gmeiner Birgitta M. Wöhrl |
| author_facet | Vanessa Boritzki Harald Hübner Anni Allikalt Peter Gmeiner Birgitta M. Wöhrl |
| author_sort | Vanessa Boritzki |
| collection | DOAJ |
| description | The human dopamine receptors D<sub>2S</sub> and D<sub>3</sub> 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 D<sub>2S</sub> and D<sub>3</sub> receptors in <i>Escherichia coli</i> 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 K<sub>D</sub>-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. |
| first_indexed | 2024-03-10T08:45:20Z |
| format | Article |
| id | doaj.art-f583f29e869f4e3d8359052d7e4dfa50 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T08:45:20Z |
| publishDate | 2021-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-f583f29e869f4e3d8359052d7e4dfa502023-11-22T07:58:26ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-08-012216864710.3390/ijms22168647Optimizing the Expression of Human Dopamine Receptors in <i>Escherichia coli</i>Vanessa Boritzki0Harald Hübner1Anni Allikalt2Peter Gmeiner3Birgitta M. Wöhrl4Department of Biochemistry IV—Biopolymers, Universität Bayreuth, Universitätsstr. 30, 95447 Bayreuth, GermanyDepartment of Chemistry and Pharmacy, Medicinal Chemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Nikolaus-Fiebiger Str. 10, 91058 Erlangen, GermanyDepartment of Chemistry and Pharmacy, Medicinal Chemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Nikolaus-Fiebiger Str. 10, 91058 Erlangen, GermanyDepartment of Chemistry and Pharmacy, Medicinal Chemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Nikolaus-Fiebiger Str. 10, 91058 Erlangen, GermanyDepartment of Biochemistry IV—Biopolymers, Universität Bayreuth, Universitätsstr. 30, 95447 Bayreuth, GermanyThe human dopamine receptors D<sub>2S</sub> and D<sub>3</sub> 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 D<sub>2S</sub> and D<sub>3</sub> receptors in <i>Escherichia coli</i> 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 K<sub>D</sub>-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.https://www.mdpi.com/1422-0067/22/16/8647human dopamine receptorsexpression in <i>E. coli</i>GPCRprotein engineeringFACSradioligand binding |
| spellingShingle | Vanessa Boritzki Harald Hübner Anni Allikalt Peter Gmeiner Birgitta M. Wöhrl Optimizing the Expression of Human Dopamine Receptors in <i>Escherichia coli</i> International Journal of Molecular Sciences human dopamine receptors expression in <i>E. coli</i> GPCR protein engineering FACS radioligand binding |
| title | Optimizing the Expression of Human Dopamine Receptors in <i>Escherichia coli</i> |
| title_full | Optimizing the Expression of Human Dopamine Receptors in <i>Escherichia coli</i> |
| title_fullStr | Optimizing the Expression of Human Dopamine Receptors in <i>Escherichia coli</i> |
| title_full_unstemmed | Optimizing the Expression of Human Dopamine Receptors in <i>Escherichia coli</i> |
| title_short | Optimizing the Expression of Human Dopamine Receptors in <i>Escherichia coli</i> |
| title_sort | optimizing the expression of human dopamine receptors in i escherichia coli i |
| topic | human dopamine receptors expression in <i>E. coli</i> GPCR protein engineering FACS radioligand binding |
| url | https://www.mdpi.com/1422-0067/22/16/8647 |
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