Utilizing the power of synthetic biology and De Novo design for the overexpression and biochemical stabilization of KCNA6 or Kv1.6 potassium channels in the E. coli expression system

利用合成生物学和 De Novo 设计的力量，实现大肠杆菌表达系统中 KCNA6 或 Kv1.6 钾通道的过度表达和生化稳定

• 批准号: 10666856
• 负责人: Luis Gonzalo Cuello
• 金额: $ 15.3万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10666856
• 关键词: Affinity; Alkali Metals; Applications Grants; Binding; Biochemical; Biological Assay; Biological Process; Bypass; C-terminal; Calorimetry; Cells; Central Nervous System; Chimeric Proteins; Consumption; Correlation Studies; Cryoelectron Microscopy; Crystallography; Dependence; Detergents; Differential Scanning Calorimetry; Dissociation; Electron Spin Resonance Spectroscopy; Electrophysiology (science); Enhancers; Escherichia coli; Eukaryotic Cell; Evaluation; Family; Fluorescence; Fluorescence Spectroscopy; Genes; Genetic Engineering; Homeostasis; Human; In Vitro; Insecta; Ion Channel; Ions; Kinetics; Knowledge; Laboratories; Libraries; Lipid Bilayers; Lipids; Liposomes; Mainstreaming; Mammalian Cell; Measurement; Measures; Membrane Proteins; Metal Ion Binding; Methodology; Methods; Molecular Conformation; Nervous System; Pichia; Potassium Channel; Preparation; Production; Proteins; Protocols documentation; Recombinants; Reporting; Structure; System; Temperature; Time; Titrations; Voltage-Gated Potassium Channel; X-Ray Crystallography; biophysical analysis; cell growth; cost; design; experience; inhibitor; interest; member; milligram; nervous system disorder; novel; novel strategies; overexpression; patch clamp; protein expression; protein structure; scale up; screening; synthetic biology

Project summary The expression of human membrane proteins has been monopolized by the outrageously expensive and time-consuming mammalian cell protein expression systems. This aspect is even worse for ion channels since their overexpression causes a significant unbalance of the cell homeostasis and hence their expression become toxic hampering cell growth and considerably lowering the yield of the recombinant ion channel and/or compromising their biochemical stability for downstream processing. My laboratory is highly focused in understanding how the structure determine the function of biomedically relevant human voltage gated K+- channels at the nervous system and we have ample experience using Isothermal Titration Calorimetry, Differential Scanning Calorimetry, Macromolecular Crystallography, Electrophysiology, Continuous Wave Electron Paramagnetic Resonance and Fluorescence Spectroscopy to assess the structure, energetic and kinetics of the conformational changes underlying the biological function of these ion channels. However, the mainstream methods for the recombinant overexpression of human ion channels are slow, expensive and time consuming (i.e., Pichia pastoris, insect cells and mammalian cells) and since we need to extract them, in many cases, with extremely expensive detergents, this becomes the main bottleneck during the production of large quantities of properly folded, biochemically stable, and functional ion channels. To by-pass these limitations, my laboratory has achieved high level expression of properly folded and fully functional human ion channels in E. coli by combining a Denovo Expression Enhancer Protein (DEEP), which enhances the expression levels of heterologous proteins. This new approach for the expression of human ion channels in E. coli circumvent the complexity and excessive cost of producing recombinant channels in eukaryotic cells. We will develop and adapt our methodology to overexpress, purify, functionally evaluate, and measure the ion binding affinity by Isothermal Titration Calorimetry of an understudied representative member of the voltage gated K+-channels (VGKC) superfamily such as: KCNA6 or Kv1.6, a channel of unknown experimentally determined structure and with no systematic functional characterization.

项目摘要 人膜蛋白的表达一直被昂贵的， 耗时的哺乳动物细胞蛋白表达系统。这方面对于离子通道甚至更糟，因为 它们的过表达引起细胞内稳态的显著失衡，因此它们的表达变得 毒性阻碍细胞生长并显著降低重组离子通道的产量，和/或 损害了它们在下游加工中的生物化学稳定性。我的实验室高度专注于 了解结构如何决定生物医学相关的人体电压门控K+-的功能 神经系统的通道，我们有丰富的经验，使用等温滴定量热法， 差示扫描量热法、高分子晶体学、电生理学、连续波 电子顺磁共振和荧光光谱，以评估结构，能量和 这些离子通道的生物学功能的构象变化的动力学。但 用于重组过表达人离子通道的主流方法是缓慢、昂贵和耗时的 消耗（即，巴斯德毕赤酵母（Pichia pastoris）、昆虫细胞和哺乳动物细胞），并且由于我们需要提取它们，在许多情况下， 在使用极其昂贵的洗涤剂的情况下，这成为生产大型 大量正确折叠、生化稳定和功能性离子通道。 为了克服这些限制，我的实验室已经实现了高水平的表达， 在E.通过组合Denovo表达增强蛋白（DEEP）， 其增强了异源蛋白的表达水平。这种表达人类基因的新方法 离子通道在E.大肠杆菌规避了生产重组通道的复杂性和过高的成本， 真核细胞我们将开发和调整我们的方法，以过表达，纯化，功能评估， 通过等温滴定量热法测量待研究代表性成员的离子结合亲和力 电压门控K+通道（VGKC）超家族的成员，如：KCNA 6或Kv1.6，一种未知的通道 实验确定的结构，没有系统的功能表征。