Membrane Protein Expression, Purification

膜蛋白表达、纯化

• 批准号: 10229567
• 负责人: Robert M Stroud
• 金额: $ 14.57万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-27 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10229567
• 关键词: Bacterial Proteins; Biological Assay; CD4 Antigens; Cell surface; Cells; Clathrin Adaptors; Complex; Cryoelectron Microscopy; Crystallization; Cytoplasm; Detergents; Endoplasmic Reticulum; Gene Mutation; Genes; Goals; Golgi Apparatus; HIV; HIV Receptors; HIV-1; Image; Immune response; Infection; Integral Membrane Protein; Label; Lead; Life; Lipids; Machine Learning; Membrane; Membrane Proteins; Methodology; Methods; Micelles; Pathway interactions; Phase; Phenocopy; Porifera; Problem Sets; Protein Structure Databases; Proteins; Resolution; Resources; Roentgen Rays; Sampling; Serine; Structure; System; TFAP2A gene; Thermodynamics; Ubiquitination; Viral; Viral Envelope Proteins; Virion; Virus; Virus Receptors; Work; X ray diffraction analysis; base; design and construction; nanodisk; nef Protein; novel; prevent; protein expression; receptor; thermostability; trafficking; vpu Protein

Membrane proteins (MPs) constitute a special set of problems concerning expression in a functional form, and purification to a pure, homogeneous and stable entity suitable for structural approaches using both cryo-EM (Core 3) and X-ray methods (Core 6). Less than 1% of the entries in the protein structure database are membrane proteins and most of these are bacterial proteins, reflecting the much greater difficulties of suitable expression, purification and crystallization of membrane proteins, and particularly eukaryotic membrane proteins. Among the accessory proteins in HIV is the membrane protein Vpu. Vpu interacts with several host soluble and membrane proteins including two well-characterized targets for degradation via ubiquitination pathways, these being the receptor for HIV, CD4, and BST-2 (tetherin). Mutation of the gene, selection of constructs, and expression of Vpu, and formation of complexes with both soluble and membrane proteins are therefore a high priority. Vpu also forms a pentameric viroporin structure that we prepare for structure analysis and determination in Project 3. The accessory protein Nef, together with the clathrin adaptor AP-2 and other factors, form an interaction network with host 10-crossing integral membrane proteins SERINC3, or SERINC5 to target these restriction factors for destruction. Expression of SERINC3 and 5 are a priority for the goals of Project 4, which are to define this interaction network at atomic resolution. The core will screen a variety of species and constructs to obtain the quantity and quality of protein needed for structure analysis. We have developed a general method to prioritize, express, purify and crystallize integral membrane proteins. We will express, purify, thermodynamically stabilize and produce membrane proteins (MPs) suitable for cryo-EM and/or X-ray diffraction of complexes. Prioritized complexes will be assayed as to homogeneity, purity and stability. Purification will be optimized for structure determination by cryo-EM imaging, which is particularly well suited to complexes of relatively large size, and for crystallization. Crystallization will use detergent-lipid screens, bicelles, nanodiscs, cubic and sponge phase crystallizations (Core 6).

膜蛋白(MPS)构成了一组与功能形式的表达有关的特殊问题，并且 纯化为纯净、均匀和稳定的实体，适用于使用低温EM的结构方法 (核心3)和X射线方法(核心6)。蛋白质结构数据库中只有不到1%的条目是 膜蛋白，其中大部分是细菌蛋白，反映了适应的难度更大 膜蛋白，特别是真核膜蛋白的表达、纯化和结晶 蛋白质。在HIV的辅助蛋白中，有一种是膜蛋白VPU。VPU与多台主机交互 包括两个通过泛素化降解的具有良好特性的靶标的可溶性和膜蛋白 这些途径是HIV、CD4和BST-2(Tetherin)的受体。基因的突变，选择 VPU的构建、表达以及与可溶性蛋白和膜蛋白形成复合体的研究 因此，这是一个高度优先的问题。VPU还形成五聚体病毒孔蛋白结构，我们为结构分析做准备 和项目3中的确定。 辅助蛋白Nef与网状蛋白接头AP-2和其他因子形成相互作用 与宿主10交叉的整膜蛋白SERINC3或SERINC5形成的针对这些限制的网络 造成破坏的因素。SERINC3和5的表达是项目4目标的优先事项，即 以原子分辨率定义这个交互网络。核心将筛选各种物种和结构，以 获得结构分析所需的蛋白质的数量和质量。我们已经开发出一种通用的方法 筛选、表达、纯化和结晶完整的膜蛋白。我们将表达、净化、 热力学稳定和产生适合低温EM和/或X射线的膜蛋白 络合物的衍射。按优先顺序排列的络合物将被分析为均一性、纯度和稳定性。 将通过低温电磁成像对纯化进行优化，以确定结构，这特别适合 较大尺寸的络合物，用于结晶。结晶将使用洗涤剂-类脂屏蔽物， 二重晶胞、纳米盘、立方相和海绵相结晶(核心6)。