NGLP-1R's Prokaryotic Expression And Phage Display Peptide Screening

Many years research results have suggested that insulin resistance and functional impairment of islet beta-cell are two main causes of type II diabetes mellitus, and dysfunction of islet beta-cell may be the center of type II diabetes mellitus. In other word, insulin resistance is an initiating factor of type II diabetes mellitus, and whether islet beta-cell is normal or abnormal is the determinative factor. Insulin resistance makes type II diabetes mellitus happen. But if islet beta-cell can play its function normally and keep its compensating function, type II diabetes mellitus will not happen. Once the compensating function decreasing, type II diabetes mellitus will happen. Glucagon like peptide-1 receptor (GLP-1R) belongs to GPCR family. When our bodys ingest nutrient substances, endocrine cells of gut will release GLP-1. GLP-1 can increase insulin secreting, inhibit glucagon generating, and regulate ingestion of nutrient substances by binding GLP-1R. So, GLP-1R is an important factor in our bodys to regulate the function of islet beta-cell. Exendin-4 is a kind of nature peptide secreting by the glands of gila monster that is living at American southwest. Exendin-4 can promote islet beta-cell proliferating and inhibit islet beta-cell from apoptosis. So exendin-4 can recover and increase the function and quantity of islet beta-cell. It has been confirmed that Exendin-4 educed its above-mentioned functions by binding GLP-1R and stimulated islet beta-cell generating cAMP. Then that initiate a series of cell signal transduction to stimulate islet beta-cell secreting insulin and decreasing blood glucose.The objective of this article is to get high affinity 12 peptide by screening phage display peptide library and then provide some structure informations for designing effective antidiabetic drug. In this article, we first expressed nGLP-1R in E.coli BL21 (DE3) and purified it by affinity chromatograph and native-PAGE, then identified nGLP-1R's activity by western blotting and ligand-receptor binding assay. In the found of above-mentioned assay, we used phage display peptide library technology to screening 12 peptide by targeting nGLP-1R protein. Our reseach results showed that: (1) We got highly purified nGLP-1R/Trx and nGLP-1R with good activity. (2) We have screened and got nine homologous sequences and two of them had high affinity with nGLP-1R. We synthesized the two sequences, purified and identified them by HPLC,MS. (3) The two sequences had high affinity with nGLP-1R, but they didn't have proliferating activity to increase islet beta-cell quantity. This suggested that they didn't have any stimulation activaty to GLP-1R. But this gave us some experience bases to design more useful hypoglycemic drugs.