Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA, United States; Biochemistry and Molecular Biophysics Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.
Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA, United States.
Methods Enzymol. 2021;653:49-74. doi: 10.1016/bs.mie.2021.02.007. Epub 2021 Mar 22.
The transient receptor potential (TRP) vanilloid 2 (TRPV2) and TRP vanilloid 5 (TRPV5) cation channels play an important role in various physiological and pathophysiological processes. The heterologous expression and purification of these channels is critical for functional and structural characterization of these important proteins. Full-length rat TRPV2 and rabbit TRPV5 can both be expressed in Saccharomyces cerevisiae and affinity purified using the 1D4 epitope and antibody to yield pure, functional channels. Further, these channels can be reconstituted into lipid nanodiscs for a more functionally relevant environment. Presented here are protocols for the expression of full-length rat TRPV2 and rabbit TRPV5 in Saccharomyces cerevisiae, their affinity purification, and their reconstitution into nanodiscs for structural and functional studies.
瞬时受体电位 (TRP) 香草素 2 (TRPV2) 和 TRP 香草素 5 (TRPV5) 阳离子通道在各种生理和病理生理过程中发挥重要作用。这些通道的异源表达和纯化对于这些重要蛋白质的功能和结构特征的研究至关重要。全长大鼠 TRPV2 和兔 TRPV5 均可在酿酒酵母中表达，并使用 1D4 表位和抗体进行亲和纯化，以获得纯的、功能正常的通道。此外，这些通道可以重构成脂质纳米盘，以获得更相关的功能环境。本文提供了全长大鼠 TRPV2 和兔 TRPV5 在酿酒酵母中的表达、亲和纯化以及在纳米盘中的重建用于结构和功能研究的方案。
