Production of Recombinant Transmembrane Proteins from Mammalian Cells for Biochemical and Structural Analyses.
Production of Recombinant Transmembrane Proteins from Mammalian Cells for Biochemical and Structural Analyses.
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从哺乳动物细胞生产重组跨膜蛋白用于生化和结构分析。
DOI：
10.1002/cpcb.106
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发表时间：
2020
影响因子：
--
通讯作者：
Banerjee,Anirban
中科院分区：
文献类型：
--
作者：
Puthenveetil,Robbins;Lee,Chul-Jin;Banerjee,Anirban
Eukaryotic integral membrane proteins are key components of various biological processes. Because they are implicated in multiple diseases, it is important to understand their mechanism of action by elucidating their structure and function. Complex technical challenges associated with the generation of recombinant membrane proteins severely impair our ability to understand them using structural and biochemical methods. Here, we provide a detailed procedure to address and mitigate difficulties involved in the large‐scale heterologous overexpression and purification of eukaryotic membrane proteins using HEK293S GnTi−cells transduced with baculovirus. Two human proteins, hDHHC15 and hPORCN, are presented as examples, with step‐by‐step instructions for transient transfection and generation of baculoviruses, followed by overexpression and purification from HEK293S GnTi−cells. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Small‐scale protein expression in mammalian HEK293T cellsBasic Protocol 2: Generation of baculovirus from Sf9 (insect) cellsAlternate Protocol: Enumeration‐free method for generating P2viral stockSupport Protocol 1: Small‐scale transduction of HEK293T cells with P2baculovirusBasic Protocol 3: Large‐scale viral transduction of HEK293S GnTi−cellsSupport Protocol 2: Large‐scale membrane preparation from HEK293S GnTi−cellsBasic Protocol 4: Large‐scale purification of membrane proteins from HEK293S GnTi−cells
