Affinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin B receptor

Abstract The development of simple molecular assays with membrane protein receptors in a native conformation still represents a challenging task. Exosomes are extracellular vesicles which, due to their stability and small size, are suited for analysis in various assay formats. Here, we describe a no...

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Autores principales:	Richard Desplantes, Christian Lévêque, Benjamin Muller, Manuela Lotierzo, Géraldine Ferracci, Michel Popoff, Michael Seagar, Robert Mamoun, Oussama El Far
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/9910d540c8b94f5dbe7a7370c996bd77
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spelling	oai:doaj.org-article:9910d540c8b94f5dbe7a7370c996bd772021-12-02T16:08:21ZAffinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin B receptor10.1038/s41598-017-01198-12045-2322https://doaj.org/article/9910d540c8b94f5dbe7a7370c996bd772017-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-01198-1https://doaj.org/toc/2045-2322Abstract The development of simple molecular assays with membrane protein receptors in a native conformation still represents a challenging task. Exosomes are extracellular vesicles which, due to their stability and small size, are suited for analysis in various assay formats. Here, we describe a novel approach to sort recombinant fully native and functional membrane proteins to exosomes using a targeting peptide. Specific binding of high affinity ligands to the potassium channel Kv1.2, the G-protein coupled receptor CXCR4, and the botulinum neurotoxin type B (BoNT/B) receptor, indicated their correct assembly and outside out orientation in exosomes. We then developed, using a label-free optical biosensor, a new method to determine the kinetic constants of BoNT/B holotoxin binding to its receptor synaptotagmin2/GT1b ganglioside (kon = 2.3 ×105 M−1.s−1, koff = 1.3 10−4 s−1), yielding an affinity constant (KD = 0.6 nM) similar to values determined from native tissue. In addition, the recombinant binding domain of BoNT/B, a potential vector for neuronal delivery, bound quasi-irreversibly to synaptotagmin 2/GT1b exosomes. Engineered exosomes provide thus a novel means to study membrane proteins for biotechnology and clinical applications.Richard DesplantesChristian LévêqueBenjamin MullerManuela LotierzoGéraldine FerracciMichel PopoffMichael SeagarRobert MamounOussama El FarNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-11 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Richard Desplantes Christian Lévêque Benjamin Muller Manuela Lotierzo Géraldine Ferracci Michel Popoff Michael Seagar Robert Mamoun Oussama El Far Affinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin B receptor
description	Abstract The development of simple molecular assays with membrane protein receptors in a native conformation still represents a challenging task. Exosomes are extracellular vesicles which, due to their stability and small size, are suited for analysis in various assay formats. Here, we describe a novel approach to sort recombinant fully native and functional membrane proteins to exosomes using a targeting peptide. Specific binding of high affinity ligands to the potassium channel Kv1.2, the G-protein coupled receptor CXCR4, and the botulinum neurotoxin type B (BoNT/B) receptor, indicated their correct assembly and outside out orientation in exosomes. We then developed, using a label-free optical biosensor, a new method to determine the kinetic constants of BoNT/B holotoxin binding to its receptor synaptotagmin2/GT1b ganglioside (kon = 2.3 ×105 M−1.s−1, koff = 1.3 10−4 s−1), yielding an affinity constant (KD = 0.6 nM) similar to values determined from native tissue. In addition, the recombinant binding domain of BoNT/B, a potential vector for neuronal delivery, bound quasi-irreversibly to synaptotagmin 2/GT1b exosomes. Engineered exosomes provide thus a novel means to study membrane proteins for biotechnology and clinical applications.
format	article
author	Richard Desplantes Christian Lévêque Benjamin Muller Manuela Lotierzo Géraldine Ferracci Michel Popoff Michael Seagar Robert Mamoun Oussama El Far
author_facet	Richard Desplantes Christian Lévêque Benjamin Muller Manuela Lotierzo Géraldine Ferracci Michel Popoff Michael Seagar Robert Mamoun Oussama El Far
author_sort	Richard Desplantes
title	Affinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin B receptor
title_short	Affinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin B receptor
title_full	Affinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin B receptor
title_fullStr	Affinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin B receptor
title_full_unstemmed	Affinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin B receptor
title_sort	affinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin b receptor
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/9910d540c8b94f5dbe7a7370c996bd77
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Affinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin B receptor

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