ArticleProceedings of the National Academy of Sciences of the United States of America2008
Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2008. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 103 papers.
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Who cites it
103 citing papers in PubMed, 197 citations in OpenAlex.
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- A Vaccinia-based system for directed evolution of GPCRs in mammalian cells.Nature communications · 2023Article
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- Generation of a Polyclonal Antibody against the Mouse Metal Transporter ZIP8.Antibodies (Basel, Switzerland) · 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
- Probing the Conformation States of Neurotensin Receptor 1 Variants by NMR Site-Directed Methyl Labeling.Chembiochem : a European journal of chemical biology · 2021Article
43 more citing papers are in PubMed but not listed here.
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Authors and funding
7 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
We outline a powerful method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli. For a mammalian G protein-coupled receptor, we arrived at a sequence with an order-of-magnitude increase in functional expression that still retains the biochemical properties of wild type. This mutant also shows enhanced heterologous expression in eukaryotes (12-fold in Pichia pastoris and 3-fold in HEK293T cells) and greater stability when solubilized and purified, indicating that the biophysical properties of the protein had been under the pressure of selection. These improvements arise from multiple small contributions, which would be difficult to assemble by rational design. In a second screen, we rapidly pinpointed a single amino acid substitution in wild type that abolishes antagonist binding while retaining agonist-binding affinity. These approaches may alleviate existing bottlenecks in structural studies of these targets by providing sufficient quantities of stable variants in defined conformational states.
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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.