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
  • [1] BIOTIN IN-VITRO TRANSLATION, NONRADIOACTIVE DETECTION OF CELL-FREE SYNTHESIZED PROTEINS
    HOELTKE, HJ
    ETTL, I
    STROBEL, E
    LEYING, H
    ZIMMERMANN, M
    ZIMMERMANN, R
    BIOTECHNIQUES, 1995, 18 (05) : 902 - &
  • [2] In vitro human cell-free expression system for synthesis of malaria proteins
    R. Yadavalli
    C. Ledger
    T. Y. Sam-Yellowe
    Parasitology Research, 2012, 111 : 2461 - 2465
  • [3] In vitro human cell-free expression system for synthesis of malaria proteins
    Yadavalli, R.
    Ledger, C.
    Sam-Yellowe, T. Y.
    PARASITOLOGY RESEARCH, 2012, 111 (06) : 2461 - 2465
  • [4] Expression of bacterial mechanosensitive channel with cell-free system
    不详
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY, 2004, 139 (04): : 777 - 777
  • [5] Functional and Structural Analysis of Cell-Free Synthesized Membrane Proteins
    Gessesse, Belay
    Ueda, Takuya
    Shimizu, Yoshihiro
    BIOPHYSICAL JOURNAL, 2019, 116 (03) : 175A - 175A
  • [6] Performance of Cell-Free Systems with Channel Reciprocity Errors
    Duarte, Rafael
    Alencar, Marcelo
    Lopes, Waslon
    Carvalho, Fabricio
    Queiroz, Wamberto
    Almeida, Danilo
    RADIOENGINEERING, 2021, 30 (01) : 237 - 249
  • [7] Downlink channel estimation in FDD cell-free massive MIMO
    Han, Tongzhou
    Zhao, Danfeng
    PHYSICAL COMMUNICATION, 2022, 52
  • [8] Assembly of Cell-Free Synthesized Ion Channel Molecules in Artificial Lipid Bilayer Observed by Atomic Force Microscopy
    Goh, Melvin Wei Shern
    Tozawa, Yuzuru
    Tero, Ryugo
    MEMBRANES, 2023, 13 (11)
  • [9] Reconstitution of the Cpx signaling system from cell-free synthesized proteins
    Miot, Marika
    Betton, Jean-Michel
    NEW BIOTECHNOLOGY, 2011, 28 (03) : 277 - 281
  • [10] Transfer mechanism of cell-free synthesized membrane proteins into mammalian cells
    Umbach, Simon
    Levin, Roman
    Neumann, Sebastian
    Steinmetzer, Torsten
    Doetsch, Volker
    Bernhard, Frank
    FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, 2022, 10