Scientific Reports (Apr 2017)

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

  • Richard Desplantes,
  • Christian Lévêque,
  • Benjamin Muller,
  • Manuela Lotierzo,
  • Géraldine Ferracci,
  • Michel Popoff,
  • Michael Seagar,
  • Robert Mamoun,
  • Oussama El Far

DOI
https://doi.org/10.1038/s41598-017-01198-1
Journal volume & issue
Vol. 7, no. 1
pp. 1 – 11

Abstract

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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.