Islet Neogenesis Associated Protein-Related Protein: From Gene to Folded Protein

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Kulis, Michael D., Jr.
Shuker, Suzanne B.
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Type 1 diabetes is the direct result of an autoimmune attack on the pancreatic islet cells. The islets contain b cells, which are the only type of cell capable of supplying insulin in the human body. The destruction of these cells leaves the diabetic to rely on exogenous insulin to maintain a normal blood sugar level. Insulin therapy allows the diabetic to deal with the symptoms of the disease, but does nothing for the underlying condition. In order to truly cure the disease, the strategy is to replenish the b cells in the diabetic. Islet neogenesis associated protein (INGAP) has been shown to regenerate islet cells and reverse experimentally-induced diabetes in animal models. The INGAP pentadecapeptide is a 15 amino acid peptide from INGAP with comparable activity to the full-length protein. This 15-mer is undergoing clinical trials for treating diabetes. The overall goal of the project described in this work is to determine the structure of the INGAP pentadecapeptide for use in structure-based drug design of non-peptide mimics of the 15-mer. The first set of experiments in the present work directly examined the 15-mer in solution using NMR. No stable structure of the small peptide was found. The second set of experiments involved a homolog of INGAP, called INGAP-related protein, or INGAPrP. INGAPrP was recombinantly produced in E. coli and subsequently purified and refolded. Refolding of INGAPrP was verified by a 1H-15N HSQC experiment. CD experiments supported the NMR study, indicating helical content in INGAPrP. The folded nature of the protein will allow for the three-dimensional structure of INGAPrP to be determined. The protein structure will show the fold of the 15-mer within the full-length protein. This information will be valuable for the ultimate goal of producing structural mimics of the INGAP pentadecapeptide. Non-peptide mimics should have better oral bioavailability and longer half-lives in vivo.
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