ArticleScientific reports2016
A generic selection system for improved expression and thermostability of G protein-coupled receptors by directed evolution.
Article in Scientific reports, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed.
- Opportunities and challenges in design and optimization of protein function.Nature reviews. Molecular cell biology · 2024Review
- Target-based drug discovery: Applications of fluorescence techniques in high throughput and fragment-based screening.Heliyon · 2024Review
- A Vaccinia-based system for directed evolution of GPCRs in mammalian cells.Nature communications · 2023Article
- Universal platform for the generation of thermostabilized GPCRs that crystallize in LCP.Nature protocols · 2022Review
- Optimizing the Expression of Human Dopamine Receptors inInternational journal of molecular sciences · 2021Article
- Directed evolution for high functional production and stability of a challenging G protein-coupled receptor.Scientific reports · 2021Article
- Engineering of Challenging G Protein-Coupled Receptors for Structure Determination and Biophysical Studies.Molecules (Basel, Switzerland) · 2021Review
- IMPROvER: the Integral Membrane Protein Stability Selector.Scientific reports · 2020Article
- Advances in G protein-coupled receptor high-throughput screening.Current opinion in biotechnology · 2020Review
- Dynamics of Co-translational Membrane Protein Integration and Translocation via the Sec Translocon.Journal of the American Chemical Society · 2020Review
- Expression and purification of recombinant G protein-coupled receptors: A review.Protein expression and purification · 2020Review
- DNA-encoded chemical libraries - achievements and remaining challenges.FEBS letters · 2018Review
- Carbohydrate active enzyme domains from extreme thermophiles: components of a modular toolbox for lignocellulose degradation.Extremophiles : life under extreme conditions · 2018Review
- Machine learning to design integral membrane channelrhodopsins for efficient eukaryotic expression and plasma membrane localization.PLoS computational biology · 2017Article
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4 authors.
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Abstract
Structural and biophysical studies as well as drug screening approaches on G protein-coupled receptors (GPCRs) have been largely hampered by the poor biophysical properties and low expression yields of this largest class of integral membrane proteins. Thermostabilisation of GPCRs by introduction of stabilising mutations has been a key factor to overcome these limitations. However, labelled ligands with sufficient affinity, which are required for selective binding to the correctly folded receptor, are often not available. Here we describe a novel procedure to improve receptor expression and stability in a generic way, independent of specific ligands, by means of directed evolution in E. coli. We have engineered a homogenous fluorescent reporter assay that only detects receptors which are correctly integrated into the inner cell membrane and, thus, discriminates functional from non-functional receptor species. When we combined this method with a directed evolution procedure we obtained highly expressing mutants of the neurotensin receptor 1 with greatly improved thermostability. By this procedure receptors with poor expression and/or low stability, for which no ligands or only ones with poor binding properties are available, can now be generated in quantities allowing detailed structural and biophysical analysis.
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