High-yield production and characterization of functional Kir2.1 ion channel using 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 cell-free protein expression system (CFPS) to synthesize Kir2.1 in the presence of peptide surfactant AK, 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.