Real time analysis of intact organelles using surface plasmon resonance

Ferracci, Géraldine; Seagar, Michael; Courageot, Joël; Miquelis, Raymond; Lévêque, Christian

doi:10.1016/j.ab.2004.08.002

Cited by 16 publications

(20 citation statements)

References 42 publications

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“…SYT2 would thus represent 3.6–7.2% of total proteins in SYT2 exosomes, depending on whether the mAb/SYT2 interaction is monovalent or bivalent. A comparison of this value with our previous results obtained in similar conditions with synaptic vesicles 22 suggests that each exosome contains about 200 copies of exogenously expressed protein (see supplementary Methods).…”

Section: Resultssupporting

confidence: 67%

“…We found 0.07 RU (resonane units) of specific mAb/RU of immobilized exosomes for exoSYT2 and 0.1RU/RU for exoCXCR4, yielding a mean of 0.08 +/− 0.01 RU (n = 4) of bound antibody per RU of immobilized exosomes (not shown). Assuming that 1RU of mAb bound = 1 pg/mm 2 of molecule 22 on the exosome surface, a protein to lipid ratio of 2:1 (w:w) and that the molecular weight of SYT2/DCTM is 53 000 Da, one can calculate a content of 0.7–1.4 nmoles of SYT2/mg of protein. SYT2 would thus represent 3.6–7.2% of total proteins in SYT2 exosomes, depending on whether the mAb/SYT2 interaction is monovalent or bivalent.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

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

Desplantes

Lévêque

Müller

et al. 2017

Sci Rep

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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.

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Section: Resultssupporting

confidence: 67%

Section: Resultsmentioning

confidence: 99%

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

Desplantes

Lévêque

Müller

et al. 2017

Sci Rep

Self Cite

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show abstract

“…For example, Biacore chips can be used not only to immobilise liposomes prepared from the synthetic lipids, but also other vesicular preparations from cells (Kim et al, 2004). Just recently Ferracci et al (2004) immobilised pure intact synaptic vesicles on the surface of the Biacore sensor chip. Such preparations enable the study of intact membrane proteins, i.e.…”

Section: Perspectivesmentioning

confidence: 99%

Surface plasmon resonance in protein–membrane interactions

Beseničar

Maček

Lakey

et al. 2006

Chemistry and Physics of Lipids

207

142

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“…This technology is based on the detection of changes in mass concentration at a biospecific interface [18]. Till now, it has been widely used in many fields, from biotechnology, medical diagnosis, drug screening, and environment protection to food quality control, due to its advantages as label-free detection, real-time monitoring, fast sensing and small sample volume [19][20][21][22][23][24].…”

Section: Introductionmentioning

confidence: 99%