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Directed evolution of class B G-protein coupled receptors

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Directed evolution for improved drug targeting

To develop a new drug one needs to know the detailed molecular structure of a targeted protein. An EU study aimed to optimise heterologous protein expression to obtain a more stable protein needed for a structural study.


Class B G-protein coupled receptors (GPCRs) are peptide hormone-binding receptors involved in several human diseases, which makes them important potential targets for drug therapy. A detailed understanding of their molecular structure, attainable through X-ray crystallography or nuclear magnetic resonance spectroscopy, requires large quantities of protein. As GPCRs exhibit low endogenous expression and are very unstable in solution, finding improved expression conditions for these proteins is necessary. The EU-funded GPCR EVOLUTION (Directed evolution of class B G-protein coupled receptors) project aimed to optimise human class B GPCRs for improved expression and increased thermostability. Scientists used directed evolution, a combination of randomly introduced mutations and subsequent selection of the desired phenotype. Large libraries of randomised receptor genes were expressed in Escherichia coli, with functional GPCR targeted to the inner cell membrane. Flow cytometry using fluorescently labelled ligands allowed scientists to find the mutants displaying increased receptor expression levels and ligand binding. Repetitive cycles of randomisation and selection gradually increased the level of protein expression and stability. During the course of the project, a generic selection system was established to overcome restrictions caused by unfavourable ligand properties. In addition, a newly created set of expression vectors allowed stringent tuning of protein expression on the single-cell level. Such vectors would be valuable for all difficult-to-express proteins. GPCR EVOLUTION succeeded in finding a set of mutations conferring improved expression and thermostability to class B GPCRs. The mutants were characterised for ligand binding and signalling properties. The improved properties of class B GPCR mutants will allow researchers to continue with their biophysical characterisation.


Directed evolution, drug targeting, protein expression, G-protein coupled receptors, thermostability

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