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

项目总结 人膜蛋白的表达已经被异常昂贵的和 耗时的哺乳动物细胞蛋白表达系统。这一方面对离子通道来说更糟糕，因为 它们的过度表达导致细胞动态平衡的严重失衡，因此它们的表达成为 毒性阻碍细胞生长并显著降低重组离子通道和/或 在下游加工过程中会影响其生化稳定性。我的实验室高度专注于 了解该结构如何决定与生物医学相关的人类电压门控K+的功能 神经系统的通道，我们有丰富的使用等温滴定量热法的经验， 差示扫描量热法、高分子结晶学、电生理学、连续波 用电子顺磁共振和荧光光谱学方法研究其结构、能量和 这些离子通道生物功能的构象变化动力学。然而， 目前主流的人离子通道基因重组过表达方法存在速度慢、成本高、耗时长等缺点 消耗(即毕赤酵母、昆虫细胞和哺乳动物细胞)，因为我们需要从许多 情况下，用极其昂贵的洗涤剂，这成为生产过程中的主要瓶颈 数量适当折叠、生物化学稳定和功能正常的离子通道。 为了绕过这些限制，我的实验室已经实现了正确折叠和 通过结合重新表达增强蛋白(DEEP)在大肠杆菌中实现全功能的人离子通道， 从而提高了异源蛋白的表达水平。这种表达人类基因的新方法 大肠杆菌中的离子通道绕过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，以过度表达、净化、功能评估和 用等温滴定量热法测量未被研究的代表性成员的离子结合亲和力 电压门控K+通道(VGKC)超家族如：KCNA6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。

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.