Tian Meilin, Ye Shixin
Shanghai Key Laboratory of Brain Functional Genomics, East China Normal University, Shanghai, China.
INSERM U1024, CNRS UMR 8197, Ecole Normale Supérieure, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), Paris, France.
Methods Mol Biol. 2017;1677:185-197. doi: 10.1007/978-1-4939-7321-7_10.
Genetic code expansion exploiting unnatural amino acids (Uaas) is a powerful technique to create novel protein function in vivo. Here, we provide a protocol for the incorporation of two UV-sensitive crosslinking Uaas into NMDA receptors (NMDARs), a type of glutamate-gated ion channels mediating fast synaptic transmission. Through heterologous expression in Xenopus laevis oocytes, we have identified light-sensitive NMDARs of GluN2B subtype by using the two-electrode voltage electrophysiology measurement in combination with online-UV application. Immunoblotting analysis has been used to confirm inter-subunit crosslinking.
利用非天然氨基酸（Uaas）进行遗传密码扩展是一种在体内创造新蛋白质功能的强大技术。在此，我们提供了一种将两种对紫外线敏感的交联非天然氨基酸整合到NMDA受体（NMDARs）中的方案，NMDA受体是一类介导快速突触传递的谷氨酸门控离子通道。通过在非洲爪蟾卵母细胞中的异源表达，我们结合在线紫外线应用，利用双电极电压电生理测量法鉴定了GluN2B亚型的光敏NMDARs。免疫印迹分析已用于确认亚基间的交联。
