Study of natural nanovesicles carrying olfactory receptors for the development of biosensing platforms

Natural vesicles produced from genetically engineered cells with tailored membrane receptor composition are promising building blocks for sensing biodevices. This is particularly true for the case of G-protein coupled receptors (GPCRs) present in many sensing processes in cells, whose functionality crucially depends on their lipid environment. Membrane receptors are involved in a variety of biochemical pathways and therefore constitute important targets for therapy and development of new drugs. Bioanalytical platforms and binding assays, using these transmembrane receptors, for drug screening or diagnostic require building well-characterized lipid membrane arrays, acting as supports to prevent protein denaturation during biochip processing. The controlled production of natural vesicles containing GPCRs, their characterization and their reproducible deposition on surfaces are among the outstanding challenges in the road map to realize practical biomolecular devices based on GPCRs. In addition, quantification of the protein receptors in such lipid membrane arrays is a key issue in order to produce reproducible and well-characterized chips. In this thesis we present the production and characterization of membrane nanovesicles (NV) from Saccaromyces Cerevisiae containing heterologously expressed olfactory receptors - a member of the family of GPCRs. We have demonstrated that membrane fractions from yeast cells spontaneously form closed spherical nanovesicles in solution. A simple method to homogenize the size of the nanovesicles to a diameter of around 100 nm at a concentration of more than 1010 nanovesicles mL-1 is also presented. It is also showed that after a genetic engineering process the olfactory receptors of interest were well expressed in the yeast membrane. Furthermore, we report for the first time a novel immunochemical analytical approach for the quantification of transmembrane proteins (i.e. GPCR) in their natural lipid environment. The procedure allows direct determination of tagged receptors (i.e. c-myc tag) without any previous protein purification or extraction steps. The proposed approach uses monoclonal antibodies addressed against the c-myc tag, frequently used in protein expression, on a microplate-based ELISA format with high detectability. The immunochemical method quantifies this tag on proteins or bioreceptors embedded in nanovesicles with detectability in the picomolar range, using protein bioconjugates as reference standards. The applicability of the method is demonstrated through the quantification of the c-myc-olfactory receptors (ORs, c-myc-OR1740 and c-myc-OR7D4) in plasma membrane nanovesicles (NVs). We also show by direct observation with Atomic Force Microscopy that nanovesicles deposit and flatten without rupturing on glass and gold substrates following approximately a diffusive law. We show that on glass surface coverages larger than 20-25% of the substrate can be reproducibly achieved under practical nanovesicle concentrations and reasonable time scales, while keeping to the minimum the presence of background residuals coming from the nanovesicles production process. On the other hand, on functionalized gold substrates surface coverages around 10-15% were achieved. Then, the role of surface chemistry was studied showing that modification of gold substrates indicates a higher affinity of natural nanovesicles for acid modified surfaces as compared to amino or alcohol modified surfaces. Nanovesicles deposition in acid modified gold surfaces and glass have been exploited for the generation of an array of multiple nanovesicles. Present results constitute an important step in the practical realization of biosensor devices based on natural nanovesicles integrating G-protein coupled membrane receptors. When olfactory receptors are genetically expressed in closed vesicles from natural yeast membrane fractions the verification of their capability for capturing specific odorant molecules are critical for the design of artificial noses. Thus, we demonstrated by Surface Plasmon Resonance (SPR) measurements on L1 Biacore chips that the receptors were functional. Despite the fact that the expression of olfactory receptors in nanovesicles is low, a fact that is coherent with the general expression level of GPCRs proteins in cells, the integration in nanovesicles together with a careful choice of the SPR experimental conditions and data analysis allowed us to obtain a concentration-dependent SPR response vs. odorant concentration with a sensitivity of 0.5-1.8RU/micromolar. The selectivity of OR carrying NV towards its specific odorant was proved in cross-check experiments with unspecific odorant molecules and control receptors. These results constitute a proof of concept that ORs embedded in nanovesicles properly respond to odorants and definitely open the perspective to use the surface plasmon resonance technique for the detection of small odorants at concentration in the micromolar range.Vesícules naturals produïes a partir de cèl·lules modificades genèticament són prometedors components de sensat per utilitzar com a detectors en biodispositius. Això és particularment cert en el cas de receptors adjuntats a proteïna G (GPCRs) presents en molts processos cel·lulars, on la seva funcionalitat depèn estrictament del seu entorn lipídic. Els receptors de membrana estan involucrats en una gran varietat de vies bioquímiques i per tant són objectiu d’estudi per teràpia i desenvolupament de nous fàrmacs. Per tant, plataformes bioanalítiques i assajos d’unió receptor-lligand, utilitzant receptors transmembrana, requereixen la construcció de matrius de membranes lipídiques ben caracteritzades, actuant com a suport per evitar la desnaturalització de proteïnes durant el processament del bioxip. En aquesta tesi es presenta la producció i caracterització de nanovesícules de membrana (NV) provinents de cèl·lules de llevat Saccharomyces cerevisiae que contenen receptors olfactius (un membre de la família de GPCRs) heteròlogament expressats a la membrana. Hem demostrat que les fraccions de membrana, a partir de cèl·lules de llevat, en solució formen espontàniament nanovesícules esfèriques tancades. També s’ha demostrat, que després d’un procés de enginyeria genètica els receptors olfactius van ser expressats correctament a la membrana del llevat. També s’ha presentat un mètode simple per homogeneïtzar la mida de les nanovesícules. A més a més, es presenta per primer cop un nou mètode immunoquímic per la quantificació directa de les proteïnes transmembrana (GPCR) en el seu ambient lipídic natural. El mètode utilitza anticossos monoclonals en un assaig basat en ELISA amb alta detectabilitat. L’aplicació del mètode es demostra a través de la quantificació del receptors olfactius OR1740 i OR7D4 expressats en nanovesícules de membrana plasmàtica. També es presenta, mitjançant observació directa amb AFM, com les nanovesícules es depositen i s’aplanen sense trencar-se sobre substrats de vidre i or seguint la llei de difusió. Es demostra com en el cas del vidre els màxims recobriments superficials obtinguts són del 20-25% i en el cas del or funcionalitzat del 10-15%, controlant la concentració de nanovesícules, el temps de depòsit, la presència de residus procedents del procés de producció de les nanovesícules, la química de la superfície, la força iònica del medi, etc. Finalment, s’ha demostrat per SPR que els receptors expressats eren funcionals i que aquesta tècnica òptica permet la detecció de petites molècules, com són els odorants, a les concentracions en el rang micromolar. Els resultats presentats en aquesta tesis contribueixen donant un pas important a la realització de dispositius biosensors basats en nanovesícules naturals que integren receptors de membrana adjuntats a proteïna G