Evaluation of Cell-Free Synthesized Human Channel Proteins for In Vitro Channel Research

Despite channel proteins being important drug targets, studies on channel proteins remain limited, as the proteins are difficult to express and require correct complex formation within membranes. Although several in vitro synthesized recombinant channels have been reported, considering the vast diversity of the structures and functions of channel proteins, it remains unclear which classes of channels cell-free synthesis can be applied to. In this study, we synthesized 250 clones of human channels, including ion channel pore-forming subunits, gap junction proteins, porins, and regulatory subunits, using a wheat cell-free membrane protein production system, and evaluated their synthetic efficiency and function. Western blotting confirmed that 95% of the channels were successfully synthesized, including very large channels with molecular weights of over 200 kDa. A subset of 47 voltage-gated potassium ion channels was further analyzed using a planar lipid bilayer assay, out of which 80% displayed a voltage-dependent opening in the assay. We co-synthesized KCNB1 and KCNS3, a known heteromeric complex pair, and demonstrated that these channels interact on a liposome. These results indicate that cell-free protein synthesis provides a promising solution for channel studies to overcome the bottleneck of in vitro protein production.

Automated summary

In this study, a wheat cell-free membrane protein production system was used to synthetize 250 clones of human channels, including various types of channel proteins. 95% of the channels were successfully synthesized, including very large channels with molecular weights of over 200 kDa. Functional analysis revealed that the majority of the synthesized voltage-gated potassium ion channels displayed voltage-dependent opening. The results demonstrate that cell-free protein synthesis provides a promising solution for channel studies.