Use of GPI-anchored proteins to study biomolecular interactions by surface plasmon resonance.
Surface plasmon resonance is a powerful tool to examine the kinetics of cell surface receptor-ligand interactions and requires only small amounts of protein. For these studies, one component is required in highly purified form to be coupled to the biosensor surface. The second component does not need to be purified. The human high affinity receptor for immunoglobulin G, FcgammaRI, presents a problem as the receptor itself cannot readily be produced in large amounts for purification and, as there are eight potential ligands for the receptor (human IgG1-4 and mouse IgG1, 2a, 2b and 3), it is difficult to immobilise the ligand. Using a previously established method for generating GPI-anchored proteins, we have produced and captured a soluble version of FcgammaRI and shown that it retains its affinity for human IgG1 and specificity for the different IgG subclasses. In addition, we also produced and captured a GPI-anchored version of the cell adhesion molecule CD2. This system circumvents the need for extensive receptor purification and is very rapid as solubilised receptors can be transferred from the cell surface to the sensor chip in 2 h. This system may be generally applicable for biosensor studies to other type I membrane proteins, and/or naturally occurring GPI-anchored proteins, especially where the interaction between a ligand and a panel of variant receptors is to be studied.
Harrison
P T
PT
Department of Medicine and Therapeutics, The University of Glasgow, UK.
Campbell
I W
IW
Allen
J M
JM
D000818:Animals; D018801:CD2 Antigens / Q000378:metabolism; D019556:COS Cells; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D017452:Receptors, IgG / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013499:Surface Properties
D018801:CD2 Antigens; D017261:Glycosylphosphatidylinositols; D007074:Immunoglobulin G; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins