A simple method for the reconstitution of membrane proteins into giant unilamellar vesicles.
A simple method for the reconstitution of membrane protein from submicron proteoliposomes into giant unilamellar vesicles (GUVs) is presented here: This method does not require detergents, fusion peptides or a dehydration step of the membrane protein solution. In a first step, GUVs of lipids were formed by electroformation, purified and concentrated; and in a second step, the concentrated GUV solution was added to a small volume of vesicles or proteoliposomes. Material transfer from submicron vesicles and proteoliposomes to GUVs occurred spontaneously and was characterized with fluorescent microscopy and patch-clamp recordings. As a functional test, the voltage-dependent, anion-selective channel protein was reconstituted into GUVs, and its electrophysiological activity was monitored with the patch clamp. This method is versatile since it is independent of the presence of the protein, as demonstrated by the fusion of fluorescently labeled submicron vesicles and proteoliposomes with GUVs.
Varnier
Armelle
A
CEA, DSV, iRTSV, Biopuces, Grenoble, France.
Kermarrec
Frédérique
F
Blesneac
Iulia
I
Moreau
Christophe
C
Liguori
Lavinia
L
Lenormand
Jean Luc
JL
Picollet-D'hahan
Nathalie
N
D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008858:Microscopy, Phase-Contrast; D008962:Models, Theoretical; D018408:Patch-Clamp Techniques; D011510:Proteolipids / Q000378:metabolism; D053835:Unilamellar Liposomes / Q000378:metabolism*; D050994:Voltage-Dependent Anion Channels / Q000378:metabolism
D008565:Membrane Proteins; D011510:Proteolipids; D053835:Unilamellar Liposomes; D050994:Voltage-Dependent Anion Channels; C020485:proteoliposomes