High-yield production and characterization of functional Kir2.1 ion channel using E. coli cell-free protein synthesis system
Year: 2025
Type: article
Abstract: 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 Escherichia coli cell-free protein expression system (CFPS) to synthesize Kir2.1. 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. Additionally, recombinant Kir2.1-liposome 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 and characterization of ion channels.
Authors: Tianqi Zhou, Zitong Zhao, Huizi Zhang, Yunyang Song, Yifeng Yin, Fanghui Wu, Yanli Liu, Dan Xu
Institution: Shanghai Ocean University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,500
OpenAlex ID: W4410230489