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

Affinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin B receptor.
Author Information (click to view)

Desplantes R, Lévêque C, Muller B, Lotierzo M, Ferracci G, Popoff M, Seagar M, Mamoun R, El Far O,


Desplantes R, Lévêque C, Muller B, Lotierzo M, Ferracci G, Popoff M, Seagar M, Mamoun R, El Far O, (click to view)

Desplantes R, Lévêque C, Muller B, Lotierzo M, Ferracci G, Popoff M, Seagar M, Mamoun R, El Far O,

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Scientific reports 2017 04 217(1) 1032 doi 10.1038/s41598-017-01198-1

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 ×10(5) 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.

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