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

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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.