Faculty Collaboration Database

Expression, purification and structural characterization of functionally replete thrombospondin-1 type 1 repeats in a bacterial expression system. Protein Expr Purif 2011 Dec;80(2):253-9

Date

08/09/2011

Pubmed ID

21821127

Pubmed Central ID

PMC3195888

DOI

10.1016/j.pep.2011.07.010

Scopus ID

2-s2.0-80054089028 (requires institutional sign-in at Scopus site)   16 Citations

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.

Author List

Klenotic PA, Page RC, Misra S, Silverstein RL

Author

Roy L. Silverstein MD Professor in the Medicine department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Amino Acid Sequence
Cell Migration Assays
Chemotaxis
Crystallography, X-Ray
Electrophoresis, Polyacrylamide Gel
Endopeptidases
Endothelial Cells
Escherichia coli
Humans
Isopropyl Thiogalactoside
Molecular Sequence Data
Plasmids
Protein Binding
Protein Interaction Domains and Motifs
Recombinant Fusion Proteins
Thrombospondin 1