High-yield production and characterization of functional Kir2.1 ion channel using E. coli cell-free protein synthesis system

A family of inwardly-rectifying potassium (Kir) channels plays a key role in the regulation of cellular potassium (K⁺) balance, affecting muscle, nerve and immune function. Kir channels are comprised of either homologous or heterologous tetramer of Kir subunits, each of which contains two-transmembrane domains. The challenges associated with the precise biophysical characterization of Kir channels have limited our understanding of this important class of molecules. Moreover, the complex multi-transmembrane domains inherent to Kir channels present significant obstacles in producing sufficient quantities for accurate characterization, further constraining our knowledge about these channels. In this study, we selected Kir2.1 as a model molecule and utilized an <i>Escherichia coli</i> cell-free protein expression system (CFPS) to synthesize Kir2.1 in the presence of peptide surfactant A₆K, which aids in promoting the soluble production, achieving α-helical conformations, and stabilizing membrane proteins (MPs). Ni-NTA affinity chromatography column was employed to purify Kir2.1, achieving a yield of approximately 1.5 mg/mL. Circular dichroism spectroscopy (CD) measurements confirmed that the purified Kir2.1 exhibited typical α-helix structures. Microscale thermophoresis (MST) assays demonstrated the binding capability of Kir2.1 with Hydrocinnamic Acid and ML133 hydrochloride, Kir2 channel selection inhibitory. Recombinant Kir2.1-liposomes exhibited specific channel activity to K⁺ using the voltage-sensitive fluorescent dye Oxonol VI to monitor the concentration gradient-driven potassium influx. This work contributes to enhancing both the efficiency of preparation, characterization and drug high-throughput screening of ion channels.