Production of recombinant soluble human integrin alpha4beta1.
Integrin alpha4beta1 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 developing a reagent for use in high-throughput inhibitor screening assays and for crystallisation trials and subsequent structure determination, we have generated a recombinant soluble alpha4beta1 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 alpha4 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 with similar kinetics to the native molecule.
Clark
K
K
Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, UK.
Newham
P
P
Burrows
L
L
Askari
J A
JA
Humphries
M J
MJ
D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002846:Chromatography, Affinity; D004220:Disulfides / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D039041:Integrin alpha4beta1; D016023:Integrins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D016201:Receptors, Lymphocyte Homing / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D012995:Solubility; D018411:Spodoptera; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
D000911:Antibodies, Monoclonal; D004220:Disulfides; D005353:Fibronectins; D039041:Integrin alpha4beta1; D016023:Integrins; D010446:Peptide Fragments; D016201:Receptors, Lymphocyte Homing; D011993:Recombinant Fusion Proteins; D019010:Vascular Cell Adhesion Molecule-1