Localizing Ligand Binding Sites Using Overlapping Recombinant Polypeptide Sequences of Vitronectin

Vitronectin is a glycoprotein involved in many cellular processes including blood coagulation, fibrinolysis, and cell/matrix binding. It interacts with a number of macromolecules including heparin, PAI-1, and the thrombin-antithrombin complex. We have studied the interaction of full-length vitronectin and the thrombin-antithrombin complex using a Far Western method. To localize the recognition site for thrombin-antithrombin binding to vitronectin, sequences for eight overlapping recombinant polypeptide sequences of vitronectin (spanning all 459 amino acids) were cloned into the pET system. Expression in BL21(DE3)pLysS Escherichia coli has been achieved for seven of the eight 100 amino acid fragments. Seven polypeptides (amino acids 1-100, 51-150, 101-200, 151-250, 201-300, 251-350, and 351-459) have been isolated and purified using metal-chelating affinity chromatography. These fragments have been tested for interaction with the thrombin-antithrombin pair using the Far Western technique. Results indicate interaction of the complex with vitronectin between amino acids 201-300 and possibly at another site near the C-terminal. Additionally, these expressed fragments have served as helpful reagents in mapping the epitopes of three monoclonal antibodies for vitronectin.