ArticleMicrobial cell factories2012
Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae.
Article in Microbial cell factories, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 19 papers.
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19 citing papers in PubMed, 46 citations in OpenAlex.
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- Exploring GPCR conformational dynamics using single-molecule fluorescence.Methods (San Diego, Calif.) · 2024Review
- Recovery of the histamine HScientific reports · 2023Article
- A methodology for creating mutants of G-protein coupled receptors stabilized in active state by combining statistical thermodynamics and evolutionary molecular engineering.Protein science : a publication of the Protein Society · 2022Article
- Universal platform for the generation of thermostabilized GPCRs that crystallize in LCP.Nature protocols · 2022Review
- State-Targeting Stabilization of Adenosine AInternational journal of molecular sciences · 2021Article
- Structure of the dopamine DNature communications · 2020Article
- Small-scale approach for precrystallization screening in GPCR X-ray crystallography.Nature protocols · 2020Article
- New tools for high-throughput expression of fungal secretory proteins in Saccharomyces cerevisiae and Pichia pastoris.Microbial biotechnology · 2019Article
- Ligand binding to human prostaglandin E receptor EPNature chemical biology · 2019Article
- Crystal structure of the endogenous agonist-bound prostanoid receptor EP3.Nature chemical biology · 2019Article
- Micro-scale and rapid expression screening of highly expressed and/or stable membrane protein variants in Saccharomyces cerevisiae.Protein science : a publication of the Protein Society · 2016Article
- Directed evolution of G protein-coupled receptors in yeast for higher functional production in eukaryotic expression hosts.Scientific reports · 2016Article
- Large-scale production and protein engineering of G protein-coupled receptors for structural studies.Frontiers in pharmacology · 2015Review
- Proteoliposome-based selection of a recombinant antibody fragment against the human M2 muscarinic acetylcholine receptor.Monoclonal antibodies in immunodiagnosis and immunotherapy · 2014Article
- Fluorescent probe for high-throughput screening of membrane protein expression.Protein science : a publication of the Protein Society · 2013Article
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Authors and funding
11 authors at 3 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundRecent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. Therefore, the quick screening of functionally expressed stable receptor variants is vital.
resultsWe developed a platform using Saccharomyces cerevisiae for the rapid construction and evaluation of functional GPCR variants for structural studies. This platform enables us to perform a screening cycle from construction to evaluation of variants within 6-7 days. We firstly confirmed the functional expression of 25 full-length class A GPCRs in this platform. Then, in order to improve the expression level and stability, we generated and evaluated the variants of the four GPCRs (hADRB2, hCHRM2, hHRH1 and hNTSR1). These stabilized receptor variants improved both functional activity and monodispersity. Finally, the expression level of the stabilized hHRH1 in Pichia pastoris was improved up to 65 pmol/mg from negligible expression of the functional full-length receptor in S. cerevisiae at first screening. The stabilized hHRH1 was able to be purified for use in crystallization trials.
conclusionsWe demonstrated that the S. cerevisiae system should serve as an easy-to-handle and rapid platform for the construction and evaluation of GPCR variants. This platform can be a powerful prescreening method to identify a suitable GPCR variant for crystallography.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.