Abstract：Abstract: Insulin degrading enzyme (IDE) is a key protein to maintain the balance of glucose metabolism and protein homeostasis in the body, and also an important target of clinical drugs for type 2 diabetes mellitus and Alzheimer’s disease. Herein, Escherichia coli ppSUMO system expressing a wild-type mouse-derived IDE was constructed, and expression, purification and functional determination of IDE were completed. The fusion protein obtained by the ppSUMO expression system had a histone tag and ULP1 restriction site, and can be digested by ULP1 enzyme after affinity purification, becoming an unlabeled IDE. In addition, according to physicochemical properties of the enzyme, such as the isoelectric point and the relative molecular mass, two purification methods, ion exchange chromatography and gel filtration chromatography, were adopted to distinguish it from contaminated proteins in the process of protein purification. The purified wild-type IDE was detected by an activity test using the specific fluorescent substrate Mca-RPPGFSAFK(Dnp). The purity of the enzyme obtained by this method was about 90%, and it had a good inhibitory response to the known inhi-bitor myricetin. The experiments demonstrated that the enzyme obtained by this expression system had high purity and activity, laying a foundation for studying the structure and function of IDE and finding clinical drugs in vitro.
引用本文:
彭彦皓, 肖 树, 陈 丽, 姚昱祺, 张云龙, 陆昌瑞, 陈 婷. 鼠源胰岛素降解酶的原核表达及活性检测[J]. 生命科学研究, 2021, 25(4): 283-292. PENG Yan-hao, XIAO Shu, CHEN Li, YAO Yu-qi, ZHANG Yun-long, LU Chang-rui, CHEN Ting. Prokaryotic Expression and Activity Detection of Mouse Insulin Degrading Enzyme. Life Science Research, 2021, 25(4): 283-292.
