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.

项目概要 人类膜蛋白的表达已被极其昂贵和昂贵的药物所垄断。 耗时的哺乳动物细胞蛋白表达系统。这对于离子通道来说更糟，因为 它们的过度表达会导致细胞稳态的显着失衡，因此它们的表达变得 毒性阻碍细胞生长并显着降低重组离子通道的产量和/或 损害其下游加工的生化稳定性。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，我们在使用等温滴定量热法方面拥有丰富的经验， 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法评估结构、能量和 这些离子通道的生物功能背后的构象变化动力学。然而， 重组过表达人类离子通道的主流方法缓慢、昂贵且耗时 消耗（即毕赤酵母、昆虫细胞和哺乳动物细胞），并且由于我们需要提取它们，因此在许多情况下 情况下，使用极其昂贵的清洁剂，这成为大型生产过程中的主要瓶颈 大量正确折叠、生化稳定且功能齐全的离子通道。 为了绕过这些限制，我的实验室已经实现了正确折叠和 通过结合 Denovo 表达增强蛋白 (DEEP)，在大肠杆菌中实现功能齐全的人类离子通道， 其增强异源蛋白质的表达水平。这种新的人类表达方式 大肠杆菌中的离子通道避免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来过度表达、纯化、功能评估和 通过等温滴定量热法测量待研究的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，例如：KCNA6 或 Kv1.6，未知通道 实验确定的结构，没有系统的功能表征。