Study of an AMPAR auxiliary transmembrane protein, stargazin

Stargazin是AMPA型谷氨酸受体的辅助跨膜亚单位，负责快速兴奋性突触传递。本研究的具体目的是建立一个协议，以获得重组stargazin蛋白质标记的稳定同位素进行结构和相互作用的研究，使用核磁共振（NMR）光谱作为主要的调查工具。我们使用毕赤酵母进行重组表达，因为它具有表达人膜蛋白的优势，而不是原核细胞如E。杆菌对细胞培养条件、细胞株、细胞破碎条件、细胞膜蛋白提取条件等进行了优化，最终获得了纯度为4 mg/L的重组观星蛋白。本研究中建立的方案将使stargazin的NMR研究成为可能。

Stargazin is an auxiliary transmembrane subunit of the AMPA-type glutamate receptor which is responsible for fast excitatory synaptic transmission. The specific aim of this study is to establish a protocol to obtain recombinant stargazin proteins labeled with stable isotopes to pursue structural and interaction studies using nuclear magnetic resonance (NMR) spectroscopy as a principal investigation tool. We used the yeast Pichia pastoris for the recombinant expression because it has advantages to express human membrane proteins over prokaryotic cells such as E. Coli. We optimized experimental conditions, such as cell culture conditions, cell strains, cell disruption conditions, membrane protein extraction conditions from cell membranes, and finally obtained a purified recombinant stargazing with an yield of 4 mg per liter culture. The protocol established in this study will enable the NMR study of stargazin.