Expression of soluble, recombinant alphavbeta3 integrin fragments in Escherichia coli.
No prokaryotic expression of integrin alphavbeta3 has been reported so far. We report here the expression of C-terminally truncated alphavbeta3 receptors in E. coli considering the known features required for dimerization and ligand binding. The expressed protein was insoluble despite of the addition of 'solubilizers' to the culture medium. Osmotic stress conditions combined with added exogenous solutes resulted in a small part of soluble receptor. The alphavbeta3 variants were purified from inclusion bodies or from soluble cytoplasmic maltose binding protein fusions. Heterodimerization of the subunits was proved by immunoprecipitation assays. Receptor-ligand binding was found to depend on the concentration. A competition assay with RGD peptides referred to unspecific receptor-ligand interaction. The latter fact was consistent with the finding that soluble receptors did not bind on RGD peptide-coupled sepharose (GRGDSPK sepharose).
Schroeckh
V
V
Dept. Appl. Microbiol., Hans Knoell Institute for Natural Products Research, Jena, Germany. schroeck@pmail.hki-jena.de
Hortschansky
P
P
Fricke
S
S
Luckenbach
G A
GA
Riesenberg
D
D
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D002451:Cell Compartmentation; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D014644:Genetic Variation; D002479:Inclusion Bodies; D016023:Integrins / Q000096:biosynthesis* / Q000235:genetics; D008024:Ligands; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009842:Oligopeptides / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D010957:Plasmids; D019030:Receptors, Vitronectin / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012995:Solubility
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D016023:Integrins; D008024:Ligands; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009842:Oligopeptides; D019030:Receptors, Vitronectin; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C461599:maltose transport system, E coli; C047981:arginyl-glycyl-aspartic acid