葡萄糖转运蛋白GLUT1的纳米盘构建与纯化

Construction and purification of nanodiscs for glucose transporter 1

摘要:

葡萄糖转运蛋白(glucose transporters, GLUTs)是介导细胞葡萄糖被动转运的关键膜蛋白，然而其异常过表达与肿瘤Warburg效应及化疗耐药性密切相关。GLUT是复杂的多次跨膜蛋白，在制备过程中需使用去垢剂从细胞膜中提取，但去垢剂在样品中的持续存在会破坏脂质-蛋白相互作用，可能导致GLUT构象失真及功能活性丧失，严重制约其结构与转运机制研究。为消除去垢剂的影响，保证GLUT1在体外维持天然构象，本研究采用纳米盘技术，通过膜支架蛋白MSP1E3D1与磷脂自组装形成仿生膜环境，以克服传统方法的局限性。基于昆虫细胞Sf9/Bac-to-Bac系统异源表达C端His10标签融合的GLUT1，经去垢剂增溶和anti-His抗体填料亲和层析纯化后，通过自组装构建GLUT1-纳米盘复合体，并使用Ni-NTA亲和层析对重构成功的纳米盘复合体进行再次纯化。使用圆二色谱分析和动态光散射分析证明了纳米盘复合体具有正确构象和均一的粒径。传统的GLUTs制备方法将蛋白重构于胶束体系，该体系缺乏天然脂质环境导致跨膜螺旋拓扑畸变，胶束界面还会干扰配体结合位点，阻碍构效关系解析和药物筛选。本研究成功构建了GLUT1-纳米盘复合体，为膜蛋白提供了磷脂双层环境，维持了蛋白天然构象，同时还消除了去垢剂影响，为冷冻电镜解析跨膜转运动态机制、蛋白复合系统，以及后续的高通量药物筛选提供了理想平台。

Abstract:

Glucose transporters (GLUTs) are pivotal membrane proteins that facilitate the passive transport of glucose into cells. However, their aberrant overexpression is closely linked to the Warburg effect and chemotherapy resistance of tumors. GLUTs are complex multi-pass transmembrane proteins that require detergents for extraction from the cell membrane during preparation. The persistent presence of detergents in the sample can disrupt lipid-protein interactions, potentially leading to conformational distortion and functional losses of GLUTs, severely hindering the research into their structures and transport mechanisms. To eliminate detergent interference and preserve its authentic conformation, this study employs nanodisc technology and utilizes the self-assembly of the membrane scaffold protein MSP1E3D1 and phospholipids to produce a biomimetic membrane environment, thereby overcoming the limitations of conventional methods. The C-terminal His10-tagged GLUT1 was heterologously expressed in the insect cell Sf9/Bac-to-Bac system, and the GLUT1-nanodisc complex was obtained after detergent solubilization, affinity chromatography purification via anti-His antibody resin, and self-assembly. The successfully reconstituted nanodisc complex was further purified by Ni-NTA affinity chromatography. Nanodisc reconstitution produced monodisperse GLUT1 particles that retained native secondary structure, as confirmed by far-UV circular dichroism (CD) spectroscopy and dynamic light scattering (DLS). Unlike conventional detergent micelles, which lack a true lipid bilayer, distort transmembrane-helix topology, and occlude ligand-binding sites, the nanodisc platform embeds GLUT1 in a phospholipid bilayer that preserves its authentic conformation while eliminating detergent interference. The resulting GLUT1-nanodisc complex is therefore a superior scaffold for high-resolution cryo-EM structural analysis, permitting detailed interrogation of the transporter’s conformational cycle, its interactions with partner proteins, and downstream structure-guided, high-throughput drug screening.