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Production of a Bioengineered G-Protein Coupled Receptor of Human Formyl Peptide Receptor 3
G-protein coupled receptors (GPCRs) participate in a wide range of vital regulations of our physiological actions. They are also of pharmaceutical importance and have become many therapeutic targets for a number of disorders and diseases. Purified GPCR-based approaches including structural study and...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3154916/ https://www.ncbi.nlm.nih.gov/pubmed/21853070 http://dx.doi.org/10.1371/journal.pone.0023076 |
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author | Wang, Xiaoqiang Zhang, Shuguang |
author_facet | Wang, Xiaoqiang Zhang, Shuguang |
author_sort | Wang, Xiaoqiang |
collection | PubMed |
description | G-protein coupled receptors (GPCRs) participate in a wide range of vital regulations of our physiological actions. They are also of pharmaceutical importance and have become many therapeutic targets for a number of disorders and diseases. Purified GPCR-based approaches including structural study and novel biophysical and biochemical function analyses are increasingly being used in GPCR-directed drug discovery. Before these approaches become routine, however, several hurdles need to be overcome; they include overexpression, solubilization, and purification of large quantities of functional and stable receptors on a regular basis. Here we report milligram production of a human formyl peptide receptor 3 (FPR3). FPR3 comprises a functionally distinct GPCR subfamily that is involved in leukocyte chemotaxis and activation. The bioengineered FPR3 was overexpressed in stable tetracycline-inducible mammalian cell lines (HEK293S). After a systematic detergent screening, fos-choline-14 (FC-14) was selected for subsequent solubilization and purification processes. A two-step purification method, immunoaffinity using anti-rho-tag monoclonal antibody 1D4 and gel filtration, was used to purify the receptors to near homogeneity. Immunofluorescence analysis showed that expressed FPR3 was predominantly displayed on cellular membrane. Secondary structural analysis using circular dichroism showed that the purified FPR3 receptor was correctly folded with >50% α-helix, which is similar to other known GPCR secondary structures. Our method can readily produce milligram quantities of human FPR3, which would facilitate in developing human FPR as therapeutic drug targets. |
format | Online Article Text |
id | pubmed-3154916 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-31549162011-08-18 Production of a Bioengineered G-Protein Coupled Receptor of Human Formyl Peptide Receptor 3 Wang, Xiaoqiang Zhang, Shuguang PLoS One Research Article G-protein coupled receptors (GPCRs) participate in a wide range of vital regulations of our physiological actions. They are also of pharmaceutical importance and have become many therapeutic targets for a number of disorders and diseases. Purified GPCR-based approaches including structural study and novel biophysical and biochemical function analyses are increasingly being used in GPCR-directed drug discovery. Before these approaches become routine, however, several hurdles need to be overcome; they include overexpression, solubilization, and purification of large quantities of functional and stable receptors on a regular basis. Here we report milligram production of a human formyl peptide receptor 3 (FPR3). FPR3 comprises a functionally distinct GPCR subfamily that is involved in leukocyte chemotaxis and activation. The bioengineered FPR3 was overexpressed in stable tetracycline-inducible mammalian cell lines (HEK293S). After a systematic detergent screening, fos-choline-14 (FC-14) was selected for subsequent solubilization and purification processes. A two-step purification method, immunoaffinity using anti-rho-tag monoclonal antibody 1D4 and gel filtration, was used to purify the receptors to near homogeneity. Immunofluorescence analysis showed that expressed FPR3 was predominantly displayed on cellular membrane. Secondary structural analysis using circular dichroism showed that the purified FPR3 receptor was correctly folded with >50% α-helix, which is similar to other known GPCR secondary structures. Our method can readily produce milligram quantities of human FPR3, which would facilitate in developing human FPR as therapeutic drug targets. Public Library of Science 2011-08-11 /pmc/articles/PMC3154916/ /pubmed/21853070 http://dx.doi.org/10.1371/journal.pone.0023076 Text en Wang, Zhang. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Wang, Xiaoqiang Zhang, Shuguang Production of a Bioengineered G-Protein Coupled Receptor of Human Formyl Peptide Receptor 3 |
title | Production of a Bioengineered G-Protein Coupled Receptor of Human Formyl Peptide Receptor 3 |
title_full | Production of a Bioengineered G-Protein Coupled Receptor of Human Formyl Peptide Receptor 3 |
title_fullStr | Production of a Bioengineered G-Protein Coupled Receptor of Human Formyl Peptide Receptor 3 |
title_full_unstemmed | Production of a Bioengineered G-Protein Coupled Receptor of Human Formyl Peptide Receptor 3 |
title_short | Production of a Bioengineered G-Protein Coupled Receptor of Human Formyl Peptide Receptor 3 |
title_sort | production of a bioengineered g-protein coupled receptor of human formyl peptide receptor 3 |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3154916/ https://www.ncbi.nlm.nih.gov/pubmed/21853070 http://dx.doi.org/10.1371/journal.pone.0023076 |
work_keys_str_mv | AT wangxiaoqiang productionofabioengineeredgproteincoupledreceptorofhumanformylpeptidereceptor3 AT zhangshuguang productionofabioengineeredgproteincoupledreceptorofhumanformylpeptidereceptor3 |