Production of recombinant soluble human integrin alpha 4 beta 1

Integrin alpha 4 beta 1 is a major leukocyte adhesion receptor that is a ke y target for the development of anti-inflammatory therapeutics. With the du al long-term goals of de, eloping a reagent for use in high-throughput inhi bitor screening assays and for crystallisation trials and subsequent struct ure determination, we have generated a recombinant soluble alpha 4 beta 1 r eceptor, Both subunits were truncated prior to the transmembrane domains by site-directed mutagenesis and expressed using baculovirus infection of ins ect cells. The molecular weights of the recombinant subunits were as expect ed for post-translationally unmodified protein, In addition, as observed fo r the native subunit, a proportion of the alpha 4 subunit was proteolytical ly processed into two fragments. ELISA and solid phase ligand-binding assay s were performed to investigate the folding and functionality of the solubl e integrin, The data suggest that the receptor was correctly folded and tha t it bound recombinant ligands,vith similar kinetics to the native molecule . (C) 2000 Federation of European Biochemical Societies.