A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization

Handle URI:
http://hdl.handle.net/10754/620932
Title:
A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization
Authors:
Ooi, Amanda Siok Lee; Wong, Aloysius Tze ( 0000-0002-9105-5845 ) ; Esau, Luke; Lemtiri-Chlieh, Fouad ( 0000-0002-7418-2623 ) ; Gehring, Christoph A. ( 0000-0003-4355-4591 )
Abstract:
The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K+ channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.
KAUST Department:
Division of Biological and Environmental Sciences and Engineering
Citation:
Ooi A, Wong A, Esau L, Lemtiri-Chlieh F, Gehring C (2016) A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization. Front Physiol 7. Available: http://dx.doi.org/10.3389/fphys.2016.00300.
Publisher:
Frontiers Media SA
Journal:
Frontiers in Physiology
KAUST Grant Number:
BAS/1/1013-01-01
Issue Date:
19-Jul-2016
DOI:
10.3389/fphys.2016.00300
PubMed ID:
27486406
Type:
Article
ISSN:
1664-042X
Sponsors:
This research was supported by King Abdullah University of Science and Technology (KAUST) (BAS/1/1013-01-01).
Additional Links:
http://journal.frontiersin.org/article/10.3389/fphys.2016.00300/full
Appears in Collections:
Articles

Full metadata record

DC FieldValue Language
dc.contributor.authorOoi, Amanda Siok Leeen
dc.contributor.authorWong, Aloysius Tzeen
dc.contributor.authorEsau, Lukeen
dc.contributor.authorLemtiri-Chlieh, Fouaden
dc.contributor.authorGehring, Christoph A.en
dc.date.accessioned2016-10-12T07:39:21Z-
dc.date.available2016-10-12T07:39:21Z-
dc.date.issued2016-07-19en
dc.identifier.citationOoi A, Wong A, Esau L, Lemtiri-Chlieh F, Gehring C (2016) A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization. Front Physiol 7. Available: http://dx.doi.org/10.3389/fphys.2016.00300.en
dc.identifier.issn1664-042Xen
dc.identifier.pmid27486406-
dc.identifier.doi10.3389/fphys.2016.00300en
dc.identifier.urihttp://hdl.handle.net/10754/620932-
dc.description.abstractThe human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K+ channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.en
dc.description.sponsorshipThis research was supported by King Abdullah University of Science and Technology (KAUST) (BAS/1/1013-01-01).en
dc.publisherFrontiers Media SAen
dc.relation.urlhttp://journal.frontiersin.org/article/10.3389/fphys.2016.00300/fullen
dc.rightsCopyright © 2016 Ooi, Wong, Esau, Lemtiri-Chlieh and Gehring. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.en
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjectElectrophysiologyen
dc.subjectFluorescent imagingen
dc.subjectHeterologous expressionen
dc.subjectHuman embryonic kidney 293 cellsen
dc.subjectMembrane proteinsen
dc.subjectTransfectionen
dc.titleA Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterizationen
dc.typeArticleen
dc.contributor.departmentDivision of Biological and Environmental Sciences and Engineeringen
dc.identifier.journalFrontiers in Physiologyen
dc.eprint.versionPublisher's Version/PDFen
dc.contributor.institutionInstitute of Integrative Biology of the Cell, Centre National de la Recherche Scientifique, Le Commissariat à l'Energie Atomique et aux Energies Alternatives, Paris-Sud University, Gif-Sur-Yvette, Franceen
kaust.authorOoi, Amanda Siok Leeen
kaust.authorWong, Aloysius Tzeen
kaust.authorEsau, Lukeen
kaust.authorLemtiri-Chlieh, Fouaden
kaust.authorGehring, Christoph A.en
kaust.grant.numberBAS/1/1013-01-01en

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