Isolation of microdomains of caveolae and gpi-anchored proteins

Details Isolation of microdomains of caveolae and gpi-anchored proteins Isolation of microdomains of caveolae and gpi-anchored proteins

C - Chemistry, Metallurgy

07

K

C07K 16/44 (2006.01) C12N 5/078 (2010.01) A61K 9/127 (2006.01) A61K 47/48 (2006.01) C07K 16/18 (2006.01) C07K 16/28 (2006.01) C07K 17/00 (2006.01)

Patent

CA 2228604

Methods of isolating and purifying caveolae, microdomains of GPI-anchored proteins, and membranes consisting essentially of caveolae associated with microdomains of GPI-anchored proteins from endothelial cell membranes are disclosed. The methods comprise coating a luminal surface of an endothelial cell membrane with an adherent first ionic material (e.g. (cationic) colloidal silica) by perfusion from a luminal cavity adjacent to the endothelial cell membrane, forming a pellicle by contacting the first ionic material with a second ionic material, (e.g. acrylic polymer) and isolating and purifying the pellicle. The pellicle is then processed to isolate the desired cellular component. Caveolae which are substantially free of microdomains of GPI- anchored proteins; microdomains of GPI-anchored proteins which are substantially free of caveolae; and membranes consisting essentially of caveolae, microdomains of GPI-anchored proteins, and caveolae associated with the microdomains of GPI-anchored proteins; all of which are substantially free of other cellular elements are also disclosed.

Procédés d'isolement et de purification de cavéoles, microdomaines de protéines fixées sur du glycosyl-phosphatidylinositol (GPI) et membranes essentiellement composées de cavéoles associées à des microdomaines de protéines fixées sur GPI à partir de membranes cellulaires endothéliales. Ces méthodes consistent à revêtir la surface luminale d'une membrane cellulaire endothéliale d'un premier matériau ionique adhésif (silice colloïdal (cationique) par exemple) par perfusion d'une cavité luminale contigüe à la membrane cellulaire endothéliale, consituer une pellicule en soumettant ce premier matériau ionique au contact d'un deuxième matériau ionique (un polymère acrylique par exemple) et isoler et purifier cette pellicule. La pellicule est ensuite traitée pour isoler les composants cellulaires souhaités. L'invention porte également sur des cavéoles qui sont essentiellement exemptes de microdomaines de protéines fixées sur GPI; des microdomaines de protéines fixées sur GPI qui sont essentiellement exempts de cavéoles; et des membranes constituées essentiellement de cavéoles, de microdomaines de protéines fixées sur GPI et de cavéoles associées aux microdomaines de protéines fixées sur GPI; tous ces éléments étant essentiellement exempts d'autres éléments cellulaires.

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