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

被引:5
|
作者
Nishiguchi, Rei [1 ]
Tanaka, Toyohisa [1 ]
Hayashida, Jun [2 ]
Nakagita, Tomoya [1 ]
Zhou, Wei [1 ]
Takeda, Hiroyuki [1 ]
机构
[1] Ehime Univ, Proteosci Ctr, Bunkyocho 3, Matsuyama, Ehime 7908577, Japan
[2] Nissan Chem Corp, Shiraoka 1470, Shiraoka, Saitama 3490294, Japan
关键词
cell-free membrane protein synthesis; proteoliposome; voltage-gated potassium channels; planar lipid bilayer assay; heteromeric complex; protein array; DOMAIN K+ CHANNELS; ION CHANNELS; FREE TRANSLATION; TARGETS; TREK-1;
D O I
10.3390/membranes13010048
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页数:14
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