Production of recombinant soluble human integrin α4β1

Integrin alpha 4 beta 1 is a major leukocyte adhesion receptor that is a key target for the development of anti-inflammatory therapeutics. With the dual long-term goals of de, eloping a reagent for use in high-throughput inhibitor screening assays and for crystallisation trials and subsequent structure determination, we have generated a recombinant soluble alpha 4 beta 1 receptor, Both subunits were truncated prior to the transmembrane domains by site-directed mutagenesis and expressed using baculovirus infection of insect cells. The molecular weights of the recombinant subunits were as expected for post-translationally unmodified protein, In addition, as observed for the native subunit, a proportion of the alpha 4 subunit was proteolytically processed into two fragments. ELISA and solid phase ligand-binding assays were performed to investigate the folding and functionality of the soluble integrin, The data suggest that the receptor was correctly folded and that it bound recombinant ligands,vith similar kinetics to the native molecule. (C) 2000 Federation of European Biochemical Societies.