Cell-Free Synthesis and Electrophysiological Analysis of Multipass Voltage-Gated Ion Channels Tethered in Microsomal Membranes.

4区 工程技术 Q2 Biochemistry, Genetics and Molecular Biology
Yogesh Pandey, Srujan Kumar Dondapati, Doreen Wüstenhagen, Stefan Kubick
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引用次数: 0

Abstract

Cell-free protein synthesis (CFPS) has emerged as a powerful tool for the rapid synthesis and analysis of various structurally and functionally distinct proteins. These include 'difficult-to-express' membrane proteins such as large multipass ion channel receptors. Owing to their membrane localization, eukaryotic CFPS supplemented with endoplasmic reticulum (ER)-derived microsomal vesicles has proven to be an efficient system for the synthesis of functional membrane proteins. Here we demonstrate the applicability of the eukaryotic cell-free systems based on lysates from the mammalian Chinese Hamster Ovary (CHO) and insect Spodoptera frugiperda (Sf21) cells. We demonstrate the efficiency of the systems in the de novo cell-free synthesis of the human cardiac ion channels: ether-a-go-go potassium channel (hERG) KV11.1 and the voltage-gated sodium channel hNaV1.5.

束缚在微粒体膜中的多通道电压门控离子通道的无细胞合成和电生理分析。
无细胞蛋白质合成(CFPS)已成为快速合成和分析各种结构和功能不同蛋白质的强大工具。其中包括“难以表达”的膜蛋白,如大型多通道离子通道受体。由于其膜定位,补充了内质网(ER)衍生的微粒体囊泡的真核CFPS已被证明是合成功能性膜蛋白的有效系统。在这里,我们证明了基于哺乳动物中国仓鼠卵巢(CHO)和昆虫草地贪夜蛾(Sf21)细胞裂解物的真核无细胞系统的适用性。我们证明了该系统在人类心脏离子通道的从头无细胞合成中的效率:醚-a-go-go钾通道(hERG)KV11.1和电压门控钠通道hNaV1.5。
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来源期刊
Advances in biochemical engineering/biotechnology
Advances in biochemical engineering/biotechnology 工程技术-生物工程与应用微生物
CiteScore
5.70
自引率
0.00%
发文量
29
期刊介绍: Advances in Biochemical Engineering/Biotechnology reviews actual trends in modern biotechnology. Its aim is to cover all aspects of this interdisciplinary technology where knowledge, methods and expertise are required for chemistry, biochemistry, microbiology, genetics, chemical engineering and computer science. Special volumes are dedicated to selected topics which focus on new biotechnological products and new processes for their synthesis and purification. They give the state-of-the-art of a topic in a comprehensive way thus being a valuable source for the next 3 - 5 years. It also discusses new discoveries and applications.
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