Directed evolution of G protein-coupled receptors in yeast for higher functional production in eukaryotic expression hosts
| Autor: | Pascal Egloff, Christoph Klenk, Lutz Kummer, Jendrik Schöppe, Andreas Plückthun, Erik Sedlák, Matthias Hillenbrand, Janosch Ehrenmann, Gabriela Nagy-Davidescu, Marco Schütz |
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| Přispěvatelé: | University of Zurich, Plückthun, Andreas |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Saccharomyces cerevisiae Proteins Blotting Western 610 Medicine & health Saccharomyces cerevisiae Computational biology Spodoptera Biology Bioinformatics Article Receptors G-Protein-Coupled 03 medical and health sciences 0302 clinical medicine Sf9 Cells 10019 Department of Biochemistry Animals Humans Receptors Neurotensin Prokaryotic expression Receptor G protein-coupled receptor 1000 Multidisciplinary Multidisciplinary Receptors Opioid kappa Receptors Neurokinin-1 Directed evolution biology.organism_classification Recombinant Proteins Yeast 030104 developmental biology Microscopy Fluorescence 570 Life sciences biology Host adaptation Directed Molecular Evolution 030217 neurology & neurosurgery |
| Zdroj: | Scientific Reports |
| ISSN: | 2045-2322 |
| Popis: | Despite recent successes, many G protein-coupled receptors (GPCRs) remained refractory to detailed molecular studies due to insufficient production yields, even in the most sophisticated eukaryotic expression systems. Here we introduce a robust method employing directed evolution of GPCRs in yeast that allows fast and efficient generation of receptor variants which show strongly increased functional production levels in eukaryotic expression hosts. Shown by evolving three different receptors in this study, the method is widely applicable, even for GPCRs which are very difficult to express. The evolved variants showed up to a 26-fold increase of functional production in insect cells compared to the wild-type receptors. Next to the increased production, the obtained variants exhibited improved biophysical properties, while functional properties remained largely unaffected. Thus, the presented method broadens the portfolio of GPCRs accessible for detailed investigations. Interestingly, the functional production of GPCRs in yeast can be further increased by induced host adaptation. |
| Databáze: | OpenAIRE |
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