Flow-mediated on-surface reconstitution of G-protein coupled receptors for applications in surface plasmon resonance biosensors.
To facilitate biosensor studies of G-protein coupled receptors (GPCR) and other membrane proteins, reliable methods for preparation of sensor surfaces with high protein density are required. We present here a method for the easy and rapid immobilization and reconstitution of GPCR on carboxylated dextran surfaces modified with long alkyl groups. Following amine coupling of the detergent-solubilized receptor, lipid/detergent-mixed micelles were adhered as they were injected over the immobilized surface, taking advantage of the integrated flow cells. The detergent was eluted in the subsequent buffer flow and the remaining lipid formed a bilayer on the chip surface. With this procedure, rhodopsin was functionally reconstituted in a lipid environment in approximately 1 min. This method can also be used for the easy formation of pure supported lipid bilayers for use in model membrane interaction studies.
Karlsson
Olof P
OP
Department of Biochemistry and Chemistry, Biacore AB, Rapsgatan 7, SE-754 50 Uppsala, Sweden. olof.karlsson@biacore.com
Löfås
Stefan
S
D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008823:Micelles; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism*; D012243:Rhodopsin / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*; D013997:Time Factors; D015293:Transducin / Q000737:chemistry / Q000378:metabolism
D008051:Lipid Bilayers; D008823:Micelles; D011956:Receptors, Cell Surface; D012243:Rhodopsin; D020962:Heterotrimeric GTP-Binding Proteins; D015293:Transducin