Accurate c-myc-olfactory receptor quantification on characterized natural nanovesicles for biosensor applications

Natural vesicles produced from genetically engineered cells with tailored membrane receptor composition are promising building blocks for biosensing devices [1]. This is particularly true for the case of G - protein coupled receptors (GPC Rs) present in many sensing processes in cells, whose functionality crucially depends on their lipid environment. However, the controlled production of natural vesicles containing GPCRs and their GPCRs expression level are among the outstanding challenges in the road map to realize practical biomolecular devices based on GPCRs. Largely expressed in the human body, GPCRs are implicated in a wide range of disease pathways [2], including cancer [3,4], cardiomyopathy, and schizophrenia. Around 50% of the drugs on the market regulate GPCR function, 30% of which directly target GPCRs [5]. Herein we present the production and characterization of membrane nanovesicles (nanosomes) of about 100 nm diameter from Saccaromyces Cerevisiae carrying the heterologously expre ssed olfactory receptor c - myc - OR1740 [6], a member of the family of GPCRs and representative of important candidates for the development of bioelectronic noses [7]. The nanosomes size in solution was characterized by Dynamic Light Scattering (DLS), Nanopar ticle Tracking Analysis (NTA), and Cryo - Electron Microscopy. In addition, we describe a universal direct novel c - myc - GPCR quantification without the necessity of a previous protein purification step. The quantification was carried out by a highly promising strategy based on a competitive ELISA format assay using c - myc tag - bioconjugates as model conjugates that mimics the behavior of the c - myc olfactory receptor on nanovesicles. A custom made antibody (anti - c - myc) was produced giving assay detectability in t he picomolar range and allowing the quantification of the c - myc - OR proteins. The number of olfactory receptors (OR17 - 40) per nanovesicle obtained was (30 +/- 6) OR/NS. Evidences of the OR17 - 40 functionality have been obtained by Biacore (SPR) measurements. Present results constitute an important step in the practical realization of biosensor devices based on natural nanovesicles integrating G - protein coupled membrane receptors.