Cloning, Expression, Purification And Partial Characterization Of Endostatin, Mig And Angiostatin

Angiogenesis, the formation of new capillary blood vessels from pre-existing ones, is a fundamental process required for a variety of physiological and pathological processes. Under physiological conditions, such as wound healing, tissue repair and growth and development, angiogenesis is a tightly controlled process, which results from an increased production of stimulatory factors and a concomitant decrease in the inhibitors of angiogenesis. In these situations, angiognesis occurs over a limited time period and proliferating endothelial cells are rapidly returning to their normal state of quiescence. However, many disease states can arise and are maintained by the persistence of angiogenesis and the loss of normal regulatory mechanisms. These include diabetes retinopathy, arthritis and other inflammatory disorders. The growth of all solid tumors is also dependent on angiogenesis and neovasculorization has been shown to be a critical step in the dissemination and progression of metastasis.Endogenous angiogenesis inhibitors are a group of polypeptides secreted by a variety of cells or released by proteolytic cleavage of parent peptides. Angiostatin and Endostatin are two recently discovered potent endogenous angiostatic factors and both have been shown to inhibit the growth of primary tumors and tumor metastases in mice. However, it is still not clear if the putative human Endostatin has similar function to mouse endostatin. Mig is an ELR'CXC chemokine with anti-angiogenesis property but the mechanisms of its action is unkown. In the first part of the present study, putative human Endostatin was cloned and expressed in E. coli wth a polyhistidine tag. Recombinant human Endostatin expressed in E. coli was purified on metal affinity chromatography and evaluated for the ability to inhibit the growth of primary Lewis lung carcinoma in nude mice. In the second part of the study, recombinant mouse Mig, Angiostatin and Endostatin were produced in baculovirus expression system in secreted form and purified via ion exchange chromatography or affinity chromatography. These purified proteins were compared for their anti-proliferative activity on bovine capillary endothelial cells. In addition, recombinant mouse Mig was also assayed for the ability to induce...