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Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
Published on: April 1, 2016
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Yeast-based directed-evolution for high-throughput structural stabilization of G protein-coupled receptors (GPCRs)
M Meltzer1, T Zvagelsky1, U Hadad2
1Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O.B. 653, Beer-Sheva, 8410501, Israel.
Scientific Reports
|May 23, 2022
Summary
We developed a yeast screening platform to efficiently isolate stable G protein-coupled receptors (GPCRs) for drug discovery. This method overcomes challenges in membrane protein structure determination, enabling new therapeutic targets.
Area of Science:
- Biochemistry
- Structural Biology
- Drug Discovery
Background:
- G protein-coupled receptors (GPCRs) are crucial drug targets, but their instability hinders structural studies.
- Current methods for stabilizing GPCRs are inefficient and limited in scope.
Purpose of the Study:
- To develop a high-throughput yeast-based screening platform for isolating stable GPCR variants.
- To facilitate GPCR crystallization for structure elucidation and drug development.
Main Methods:
- Utilized a yeast cell wall for compartmentalization, linking GPCR phenotype to genotype.
- Screened for GPCR variants stable in short-chain detergents, essential for crystallization.
- Employed a single-step isolation process for stable receptor variants.
Main Results:
- Successfully established a yeast screening platform for efficient isolation of stable GPCRs.
- Demonstrated the feasibility of stabilizing GPCRs through targeted mutations.
- Identified a surprising plasticity in the GPCR scaffold for stabilization.
Conclusions:
- The yeast-based platform significantly enhances the discovery of stable GPCRs.
- GPCR stabilization is achievable, opening avenues for structure-based drug design.
- Modulating the C-terminus offers a potential strategy for improving GPCR stability.
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