Oriented binding of a lipid-anchored cell adhesion protein onto a biosensor surface using hydrophobic immobilization and photoactive crosslinking

The carboxymethyl-dextran surface of a biosensor instrument was modified to couple, in an active state, the lipid-anchored contact site A (csA) glycoprotein, a homophilic adhesion molecule of aggregating cells of Dictyostelium discoideum. The carboxy groups were modified by heptyl residues for hydrophobic binding of the molecule with its lipid anchor to the dextran matrix, Alternatively, the protein was fixed in a similar orientation by covalent linkage through a perfluoro-phenylazide-derived hydrophobic crosslinker. Titration of the bound csA molecules with antibodies that recognize either the native or the denatured glycoprotein verified that the csA molecules were coupled in a native state to the sensor surface, Interaction of the immobilized csA protein with csA in solution established that the bound molecules are capable of taking part in homophilic interactions. (C) 1996 Academic Press, Inc.