Directed evolution for high functional production and stability of a challenging G protein-coupled receptor

Abstract Membrane proteins such as G protein-coupled receptors (GPCRs) carry out many fundamental biological functions, are involved in a large number of physiological responses, and are thus important drug targets. To allow detailed biophysical and structural studies, most of these important recept...

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Autores principales:	Yann Waltenspühl, Jeliazko R. Jeliazkov, Lutz Kummer, Andreas Plückthun
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/d30d7a1ad5c34e6caa1d47c532bf4b8b
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spelling	oai:doaj.org-article:d30d7a1ad5c34e6caa1d47c532bf4b8b2021-12-02T17:33:00ZDirected evolution for high functional production and stability of a challenging G protein-coupled receptor10.1038/s41598-021-87793-92045-2322https://doaj.org/article/d30d7a1ad5c34e6caa1d47c532bf4b8b2021-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-87793-9https://doaj.org/toc/2045-2322Abstract Membrane proteins such as G protein-coupled receptors (GPCRs) carry out many fundamental biological functions, are involved in a large number of physiological responses, and are thus important drug targets. To allow detailed biophysical and structural studies, most of these important receptors have to be engineered to overcome their poor intrinsic stability and low expression levels. However, those GPCRs with especially poor properties cannot be successfully optimised even with the current technologies. Here, we present an engineering strategy, based on the combination of three previously developed directed evolution methods, to improve the properties of particularly challenging GPCRs. Application of this novel combination approach enabled the successful selection for improved and crystallisable variants of the human oxytocin receptor, a GPCR with particularly low intrinsic production levels. To analyse the selection results and, in particular, compare the mutations enriched in different hosts, we developed a Next-Generation Sequencing (NGS) strategy that combines long reads, covering the whole receptor, with exceptionally low error rates. This study thus gave insight into the evolution pressure on the same membrane protein in prokaryotes and eukaryotes. Our long-read NGS strategy provides a general methodology for the highly accurate analysis of libraries of point mutants during directed evolution.Yann WaltenspühlJeliazko R. JeliazkovLutz KummerAndreas PlückthunNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Yann Waltenspühl Jeliazko R. Jeliazkov Lutz Kummer Andreas Plückthun Directed evolution for high functional production and stability of a challenging G protein-coupled receptor
description	Abstract Membrane proteins such as G protein-coupled receptors (GPCRs) carry out many fundamental biological functions, are involved in a large number of physiological responses, and are thus important drug targets. To allow detailed biophysical and structural studies, most of these important receptors have to be engineered to overcome their poor intrinsic stability and low expression levels. However, those GPCRs with especially poor properties cannot be successfully optimised even with the current technologies. Here, we present an engineering strategy, based on the combination of three previously developed directed evolution methods, to improve the properties of particularly challenging GPCRs. Application of this novel combination approach enabled the successful selection for improved and crystallisable variants of the human oxytocin receptor, a GPCR with particularly low intrinsic production levels. To analyse the selection results and, in particular, compare the mutations enriched in different hosts, we developed a Next-Generation Sequencing (NGS) strategy that combines long reads, covering the whole receptor, with exceptionally low error rates. This study thus gave insight into the evolution pressure on the same membrane protein in prokaryotes and eukaryotes. Our long-read NGS strategy provides a general methodology for the highly accurate analysis of libraries of point mutants during directed evolution.
format	article
author	Yann Waltenspühl Jeliazko R. Jeliazkov Lutz Kummer Andreas Plückthun
author_facet	Yann Waltenspühl Jeliazko R. Jeliazkov Lutz Kummer Andreas Plückthun
author_sort	Yann Waltenspühl
title	Directed evolution for high functional production and stability of a challenging G protein-coupled receptor
title_short	Directed evolution for high functional production and stability of a challenging G protein-coupled receptor
title_full	Directed evolution for high functional production and stability of a challenging G protein-coupled receptor
title_fullStr	Directed evolution for high functional production and stability of a challenging G protein-coupled receptor
title_full_unstemmed	Directed evolution for high functional production and stability of a challenging G protein-coupled receptor
title_sort	directed evolution for high functional production and stability of a challenging g protein-coupled receptor
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/d30d7a1ad5c34e6caa1d47c532bf4b8b
work_keys_str_mv	AT yannwaltenspuhl directedevolutionforhighfunctionalproductionandstabilityofachallenginggproteincoupledreceptor AT jeliazkorjeliazkov directedevolutionforhighfunctionalproductionandstabilityofachallenginggproteincoupledreceptor AT lutzkummer directedevolutionforhighfunctionalproductionandstabilityofachallenginggproteincoupledreceptor AT andreaspluckthun directedevolutionforhighfunctionalproductionandstabilityofachallenginggproteincoupledreceptor
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Directed evolution for high functional production and stability of a challenging G protein-coupled receptor

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