Covalent immobilization of recombinant human alpha(v)beta(3) integrin on asolid support with retention of functionality

alpha(v)beta(3) is the major receptor mediating the attachment of osteoclas ts to bone surface and plays a critical role in bone resorption and remodel ing, Interfering with alpha(v)beta(3) binding inhibits osteoclast-mediated bone resorption, and thus demonstrates the potential utility of alpha(v)bet a(3) antagonists for therapy of osteoporosis. This report describes the gen eration of an alpha(v)beta(3) affinity column which was created to enable s creening of collections of large numbers of ligands, e.g., combinatorial li braries (previously prepared by us), to sort and identify ligands with the highest affinity for alpha(v)beta(3). We demonstrate that covalent immobili zation of the heterodimeric alpha(v)beta(3) receptor can be achieved with r etention of characteristic ligand binding properties. Human alpha(v)beta(3) was isolated from human embryonic kidney cells (HEK 293) that stably expre ss high levels of the recombinant receptor and then affinity purified to ho mogeneity. Purified alpha(v)beta(3) receptor was linked covalently to activ ated CH-Sepharose 4B beads. Quantification of immobilized functional recept or was determined by Scatchard analysis. The immobilized functional recepto r maintains binding properties similar to the membrane-embedded and soluble receptor. The immobilized receptor also can be used to select the highest affinity ligand from among a mixture of peptides which differ in their bind ing affinity, structure, and hydrophobicity, both when the peptides are loa ded in equimolar concentrations in a mixture and when the concentration of the highest affinity ligand is reduced 10-fold. Liquid chromatography-mass spectrometry was utilized to confirm selective ligand binding and to demons trate that preferential binding was not due to nonspecific hydrophobic inte ractions with immobilized alpha(v)beta(3) receptor or the affinity column. This approach may be of general use for affinity-based screening of ligands for other integrins and should enable practical screening of combinatorial libraries containing large numbers of potential ligands for the human alph a(v)beta(3) integrin receptor, including linear peptides, cyclic peptides, and peptidomimetics. (C) 1999 Academic Press.