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Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs
BACKGROUND: G protein coupled receptors (GPCRs) represent the largest family of membrane proteins in the human genome and the richest source of targets for the pharmaceutical industry. A major limitation to characterizing GPCRs has been the difficulty in developing high-level heterologous expression...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3125327/ https://www.ncbi.nlm.nih.gov/pubmed/21605442 http://dx.doi.org/10.1186/1472-6750-11-57 |
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author | Yang, Jian-Ping Cirico, Tatiana Katzen, Federico Peterson, Todd C Kudlicki, Wieslaw |
author_facet | Yang, Jian-Ping Cirico, Tatiana Katzen, Federico Peterson, Todd C Kudlicki, Wieslaw |
author_sort | Yang, Jian-Ping |
collection | PubMed |
description | BACKGROUND: G protein coupled receptors (GPCRs) represent the largest family of membrane proteins in the human genome and the richest source of targets for the pharmaceutical industry. A major limitation to characterizing GPCRs has been the difficulty in developing high-level heterologous expression systems that are cost effective. Reasons for these difficulties include inefficient transport and insertion in the plasma membrane and cytotoxicity. Additionally, GPCR purification requires detergents, which have a negative effect on receptor yields and stability. RESULTS: Here we report a detergent-free cell-free protein expression-based method to obtain pharmacologically active GPCRs in about 2 hours. Our strategy relies on the co-translational insertion of modified GPCRs into nanometer-sized planar membranes. As a model we employed an engineered β2-adrenergic receptor in which the third intracellular loop has been replaced with T4 lysozyme (β2AR -T4L). We demonstrated that nanolipoprotein particles (NLPs) are necessary for expression of active β2AR -T4L in cell-free systems. The binding specificity of the NLP- β2AR-T4L complex has been determined by competitive assays. Our results demonstrate that β2AR-T4L synthesized in vitro depends on similar oxidative conditions as those required by an in vivo-expressed receptor. CONCLUSIONS: Although the activation of β2AR-T4L requires the insertion of the T4 lysozyme sequence and the yield of that active protein limited, our results conceptually prove that cell-free protein expression could be used as a fast approach to express these valuable and notoriously difficult-to-express proteins. |
format | Online Article Text |
id | pubmed-3125327 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-31253272011-06-29 Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs Yang, Jian-Ping Cirico, Tatiana Katzen, Federico Peterson, Todd C Kudlicki, Wieslaw BMC Biotechnol Methodology Article BACKGROUND: G protein coupled receptors (GPCRs) represent the largest family of membrane proteins in the human genome and the richest source of targets for the pharmaceutical industry. A major limitation to characterizing GPCRs has been the difficulty in developing high-level heterologous expression systems that are cost effective. Reasons for these difficulties include inefficient transport and insertion in the plasma membrane and cytotoxicity. Additionally, GPCR purification requires detergents, which have a negative effect on receptor yields and stability. RESULTS: Here we report a detergent-free cell-free protein expression-based method to obtain pharmacologically active GPCRs in about 2 hours. Our strategy relies on the co-translational insertion of modified GPCRs into nanometer-sized planar membranes. As a model we employed an engineered β2-adrenergic receptor in which the third intracellular loop has been replaced with T4 lysozyme (β2AR -T4L). We demonstrated that nanolipoprotein particles (NLPs) are necessary for expression of active β2AR -T4L in cell-free systems. The binding specificity of the NLP- β2AR-T4L complex has been determined by competitive assays. Our results demonstrate that β2AR-T4L synthesized in vitro depends on similar oxidative conditions as those required by an in vivo-expressed receptor. CONCLUSIONS: Although the activation of β2AR-T4L requires the insertion of the T4 lysozyme sequence and the yield of that active protein limited, our results conceptually prove that cell-free protein expression could be used as a fast approach to express these valuable and notoriously difficult-to-express proteins. BioMed Central 2011-05-23 /pmc/articles/PMC3125327/ /pubmed/21605442 http://dx.doi.org/10.1186/1472-6750-11-57 Text en Copyright ©2011 Yang et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methodology Article Yang, Jian-Ping Cirico, Tatiana Katzen, Federico Peterson, Todd C Kudlicki, Wieslaw Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs |
title | Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs |
title_full | Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs |
title_fullStr | Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs |
title_full_unstemmed | Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs |
title_short | Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs |
title_sort | cell-free synthesis of a functional g protein-coupled receptor complexed with nanometer scale bilayer discs |
topic | Methodology Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3125327/ https://www.ncbi.nlm.nih.gov/pubmed/21605442 http://dx.doi.org/10.1186/1472-6750-11-57 |
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