CLONING, EXPRESSION AND PURIFICATION OF UROGUANYLIN

DESCRIPTION (provided by applicant): Uroguanylin (UG) is highly expressed in intestinal mucosa and is secreted into gut where it serves as an intestinal natriuretic hormone to influence fluid and ion transport. Several studies have implicated an important role of UG in the activation of a cGMP signal transduction pathway that may help to regulate the turnover of epithelial cells and maintain homeostasis in intestinal mucosa. Recently, we as well as other investigators have demonstrated that the expression of UG is dramatically suppressed in human colon polyps, tumor tissues and in colon cancer cell lines. Furthermore, we also demonstrated that the oral administration of UG inhibits formation of polyps and their progression to adenocarcinoma in Min mouse, an animal model for colon cancer studies. Although the precise mechanism is still not clear, we believe UG binds to guanylate cyclase receptors (GC-C) and stimulates intracellular production of cyclic guanosine monophosphate (cGMP), resulting in activation of cystic fibrosis transmembrane conductance regulator (CFTR). Activation of CFFR chloride channel proteins and the subsequent enhancement of transepithelial efflux of sodium (Na+), potassium (K+), chloride and water leads to loss in cell volume, and subsequently to induction of apoptosis. Taken together, these results suggest that UG can be developed as a pharmaceutical agent to treat colon cancer in human. However, the cost of chemically synthesized UG peptide has hampered further development of this peptide as a novel therapeutic agent. In Phase I of this SBIR Grant, we will clone and express UG gene in bacteria and develop a cost effective procedure for purification of the biologically active UG. The preliminary data included in this grant suggest that the cloned UG gene is expressed in E. coli to produce a properly folded and biologically active UG. However, the level of expression was poor. We will modify the cloning vector and optimize the expression level. Cost effective and scalable procedure for production and purification of recombinant peptide from E. coli will also be developed. The overall goal of the proposed study is to determine the feasibility of recombinant UG production in a cost effective manner. Accomplishment of this goal will allow us to develop a large-scale procedure for production and purification of UG, and in vivo evaluation in animal models for colon cancer, as part of a planned Phase II.