title: Expression, purification and structural characterization of functionally replete thrombospondin-1 type 1 repeats in a bacterial expression system.
abstract: The matrix glycoprotein thrombospondin-1 (TSP-1) is a prominent regulator of endothelial cells and angiogenesis. The anti-angiogenic and anti-tumorigenic properties of TSP-1 are in part mediated by the TSP-1 type 1 repeat domains 2 and 3, TSR(2,3). Here, we describe the expression and purification of human TSR(2,3) in milligram quantities from an Escherichia coli expression system. Microvascular endothelial cell migration assays and binding assays with a canonical TSP-1 ligand, histidine-rich glycoprotein (HRGP), indicate that recombinant TSR(2,3) exhibits anti-chemotactic and ligand binding properties similar to full length TSP-1. Furthermore, we determined the structure of E. coli expressed TSR(2,3) by X-ray crystallography at 2.4Å and found the structure to be identical to the existing TSR(2,3) crystal structure determined from a Drosophila expression system. The TSR(2,3) expression and purification protocol developed in this study allows for facile expression of TSR(2,3) for biochemical and biophysical studies, and will aid in the elucidation of the molecular mechanisms of TSP-1 anti-angiogenic and anti-tumorigenic activities.
authors.affiliation: Department of Cell Biology, NC10, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
authors.forename: Philip A
authors.initials: PA
authors.lastname: Klenotic
authors.forename: Richard C
authors.initials: RC
authors.lastname: Page
authors.forename: Saurav
authors.initials: S
authors.lastname: Misra
authors.forename: Roy L
authors.initials: RL
authors.lastname: Silverstein
mesh_terms: D000595:Amino Acid Sequence; D054443:Cell Migration Assays; D002633:Chemotaxis / Q000187:drug effects; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D019700:Thrombospondin 1 / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D011993:Recombinant Fusion Proteins; D019700:Thrombospondin 1; D007544:Isopropyl Thiogalactoside; D010450:Endopeptidases; C099488:TEV protease