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
相关论文
共 50 条
  • [31] Storable droplet interface lipid bilayers for cell-free ion channel studies
    Jung, Sung-Ho
    Choi, Sangbaek
    Kim, Young-Rok
    Jeon, Tae-Joon
    BIOPROCESS AND BIOSYSTEMS ENGINEERING, 2012, 35 (1-2) : 241 - 246
  • [32] Expectation Propagation for Semi-Blind Channel Estimation in Cell-Free Networks
    Zhao, Zilu
    Slock, Dirk
    2024 JOINT EUROPEAN CONFERENCE ON NETWORKS AND COMMUNICATIONS & 6G SUMMIT, EUCNC/6G SUMMIT 2024, 2024, : 553 - 557
  • [33] RIS-Aided Cell-Free Massive MIMO Systems With Channel Aging
    Shi, Enyu
    Zhang, Jiayi
    Zheng, Jiakang
    Ai, Bo
    Ng, Derrick Wing Kwan
    IEEE TRANSACTIONS ON VEHICULAR TECHNOLOGY, 2024, 73 (08) : 11487 - 11502
  • [34] Cell-free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent
    Berrier, C
    Park, KH
    Abes, S
    Bibonne, A
    Betton, JM
    Ghazi, A
    BIOCHEMISTRY, 2004, 43 (39) : 12585 - 12591
  • [35] Cell-Free Bistatic Backscatter Communication: Channel Estimation, Optimization, and Performance Analysis
    Galappaththige, Diluka
    Rezaei, Fatemeh
    Tellambura, Chintha
    Maaref, Amine
    IEEE TRANSACTIONS ON COMMUNICATIONS, 2024, 72 (10) : 6617 - 6632
  • [36] Analysis of Selective User Forwarding in Cell-Free Massive MIMO With Channel Aging
    Francis, Maria
    Mehran, Farhad
    Hari, K. V. S.
    IEEE ACCESS, 2023, 11 : 71210 - 71223
  • [37] Channel Hardening and Favorable Propagation in Cell-Free Massive MIMO With Stochastic Geometry
    Chen, Zheng
    Bjornson, Emil
    IEEE TRANSACTIONS ON COMMUNICATIONS, 2018, 66 (11) : 5205 - 5219
  • [38] Uplink Performance Analysis of Cell-Free mMIMO Systems Under Channel Aging
    Chopra, Ribhu
    Murthy, Chandra R.
    Papazafeiropoulos, Anastasios K.
    IEEE COMMUNICATIONS LETTERS, 2021, 25 (07) : 2206 - 2210
  • [39] An Improved Channel Estimation Method for Cell-Free Uplink Systems With Scalar Quantizers
    Kim, Bumjun
    Yu, Daesung
    Park, Seok-Hwan
    11TH INTERNATIONAL CONFERENCE ON ICT CONVERGENCE: DATA, NETWORK, AND AI IN THE AGE OF UNTACT (ICTC 2020), 2020, : 813 - 816
  • [40] Channel Estimation for Reconfigurable Intelligent Surface-Assisted Cell-Free Communications
    Xie, Chenfei
    Yang, Songjie
    Zhang, Zhongpei
    TRANSACTIONS ON EMERGING TELECOMMUNICATIONS TECHNOLOGIES, 2025, 36 (03):