Servicio de Información Comunitario sobre Investigación y Desarrollo - CORDIS

FP5

SPOT-NOSED Informe resumido

Project ID: IST-2001-38899
Financiado con arreglo a: FP5-IST

Method to obtain nanoliposomes containing olfactory receptors with controllable size

The membrane fraction obtained from the yeast cells expressing the ORs consists, among other things, of nanosomes whose size can be controlled.

In the initial plan of the project it was foreseen the possibility to purify the receptors from the yeast cell in order to further immobilize them on the substrates by using techniques similar to the Langmuir-Blodgett technique. However, two main results obtained inside the projected suggested that other strategies should be addressed. On the one hand it was verified that the expression level even if high it is not high enough to be subject to a standard purification process (comparable to the purification process followed inside the project for bovine rhodopsine in the first stages of the project). On the other hand, even if purification would have been possible, it was demonstrated that the formation of reconstituted films of membrane proteins on solid substrates by the Langmuir-Blodgett technique was not possible, as verified with purified samples of bovine rhodopsin inside the project.

These results suggested an alternate immobilization strategy. The one that finally was implemented implied an in-situ purification of the produced membrane fraction on previously functionalized substrates.

To this end, it was necessary to perform a thoroughly characterisation of the membrane fraction obtained from the lyses of the yeast cells expressing the OR I7.

Characterization was performed by Transmission Electron Microscopy and Atomic Force Microscopy.

The membrane fraction was prepared as detailed: Yeast cells resuspended in ice-cold lysis buffer (50mM Tris-HCl, pH 7.5, 1mM EDTA, 0.1mM PMSF, 250mM sorbitol) and the complete protease inhibitor cocktail can be disrupted by either of 2 methods:
- Vortexing with glass beads. Glass beads are added and cells are disrupted by 7 cycles of 1 min of vigorous vortexing/1 min of cooling on ice.
- Cell disintegration using a Cell Disrupter "Basic Z" (Constant System Ltd). Yeast cells can be disrupted at 4°C at a pressure of 1200 bars.

In both cases, samples are then centrifuged at 5000g for 10 min at 4°C to remove unbroken cells and cell walls. The supernatant is further centrifuged at 40,000 g for 40min at 4°C. This second pellet, enriched in plasma membranes, is resuspended in the lysis buffer with a Dounce homogeniser, and stored in aliquots at 80°C.
Negative staining electron microscopy of this membrane fraction was performed.

It is thus clear that the fragments are indeed circularised into microsomes: the bilayer is visualised at the outline of the structures. Their size ranges from hundreds of nm (large structures, microsomes) to tens of nm (small structures, nanosomes).

A further sonication of the samples was used to reduce the size of the fragments present in these membrane fractions and make it uniform (sonication bath). A number of experimental parameters should be considered: power output level, duty cycle, total sonication time.

An additional 20 minutes sonication indeed yields nanosomes of uniform size (40-60nm).

From these studies we concluded that the preparation procedures used provide membrane fraction samples mainly containing nanosomes of uniform (40-60nm) size. This size seems to be in good adaptation with the geometrical requirements of the other workpackages of the project.

Concerning Atomic Force Microscopy characterization structural characterisation of the nanosomes containing the olfactory receptors provided by WP1 once immobilised on a solid substrate and in liquid environment has been performed with the AFM set up. Experiments have been performed on both bare and functionalised gold substrates in order to check the effects of the hydrophobicity on the structural properties of the nanosomes.

The aspect ratio of the nanosomes once adsorbed is quite low (typically below 1:3). This rule is followed by a wide range of measured nanosome sizes and for the two types of surfaces analysed.

We note that effects of the hydrophobicity are not evident from the measurements performed.

Finally, we analysed the surface coverage of the adsorbed nanosomes. While on non sonicated samples the coverage is quite poor (below 10%), after sonication coverages close to the 50% have been measured on bare gold. In addition to higher surface coverage, homogenisation of the sizes around 50 nm of the nanosomes has been noticed.

Finally, we have performed Transmission Electron Microscopic images of single nanosomes to provide a more direct prove of the presence of olfactory receptors in them. Some protuberances of size comparable to a single olfactory receptor are evident on the nanosome surface, although not all of them would correspond to olfactory receptors. Some of the images obtained show a single nanosome containing OR 1740 preparaded by inmunostaining with gold nanoparticles of 5nm diameter, demonstrating that some ORs are present in the nanosome.

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INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE
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