人源γ-分泌酶的重要亚基nicastrin蛋白表达与纯化

Expression and Purification of Nicastrin, One of Essential Subunits of γ-Secretase from Homo sapiens

γ-分泌酶是具有天冬氨酸内切酶活性跨膜蛋白复合物,与阿尔茨海默症的病理特征的β-淀粉样蛋白(amyloid proteinβ,Aβ)生成有关。Nicastrin(NCT)是Ⅰ型单次跨膜糖蛋白,具有709个氨基酸。它是γ-分泌酶的重要组分,具有募集和结合γ-分泌酶底物的功能,参与γ-分泌酶的组装过程。本研究中,利用昆虫杆状病毒表达系统表达人源NCT全长和胞外结构域,得到了纯度较好的蛋白,用于晶体初筛。由于nicastrin自身高度糖基化,使用Endo H和PNGase F去糖基化酶进行体外脱糖基作用,使得蛋白均一度提高,纯化到较稳定的胞外结构域蛋白,为下一步对人源nicastrin蛋白进行结构生物学研究奠定了基础。

γ-secretase complex belongs to intramembrane-cleaving proteases family. It is related to the generation of amyloid protein β which aberrant accumulation can lead to the Alzheimer's disease. Nicastrin is a single pass type Ⅰ transmembrane glycoprotein with 709 amino acids. It is the critical component of γ-secretase complex, involved in recognition and binding the substrates and essential for the assembly of γ-secretase. In this research, we obtained the purified the human nicastrin-FL and human nicastrin extracellular domain proteins expressed in bac-to-bac baculovirus expression system. The protein with high purity and were applied for the crystal screening. The glycosylated nicastrin was de-glycosylated by Endo H and PNGase F to obtain more stable target protein. This laid a solid foundation for further crystallization trial and structural biology study.