从哺乳动物细胞中生产重组跨膜蛋白用于生化和结构分析

Q3 Biochemistry, Genetics and Molecular Biology
Robbins Puthenveetil, Chul-Jin Lee, Anirban Banerjee
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引用次数: 3

摘要

真核生物整体膜蛋白是各种生物过程的关键组成部分。由于它们与多种疾病有关,因此通过阐明它们的结构和功能来了解它们的作用机制非常重要。与重组膜蛋白产生相关的复杂技术挑战严重损害了我们使用结构和生化方法理解它们的能力。在这里,我们提供了一个详细的程序,以解决和减轻困难涉及大规模外源过表达和纯化真核膜蛋白用HEK293S GnTi -细胞转导杆状病毒。以hDHHC15和hPORCN两种人蛋白为例,逐步指导瞬时转染和杆状病毒的产生,然后从HEK293S GnTi−细胞中过表达和纯化。©2020 Wiley期刊有限公司基本方案1:哺乳动物HEK293T细胞的小范围蛋白表达基本方案2:从Sf9(昆虫)细胞中生成杆状病毒替代方案:无计数法生成P2病毒储备支持方案1:HEK293T细胞与P2杆状病毒的小范围转导基本方案3:HEK293S GnTi -细胞的大规模病毒转导支持方案2:HEK293S GnTi -细胞的大规模膜制备基本方案4:HEK293S GnTi−细胞膜蛋白的大规模纯化
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Production of Recombinant Transmembrane Proteins from Mammalian Cells for Biochemical and Structural Analyses

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 cells

Basic Protocol 2: Generation of baculovirus from Sf9 (insect) cells

Alternate Protocol: Enumeration-free method for generating P2 viral stock

Support Protocol 1: Small-scale transduction of HEK293T cells with P2 baculovirus

Basic Protocol 3: Large-scale viral transduction of HEK293S GnTi cells

Support Protocol 2: Large-scale membrane preparation from HEK293S GnTi cells

Basic Protocol 4: Large-scale purification of membrane proteins from HEK293S GnTi cells

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来源期刊
Current Protocols in Cell Biology
Current Protocols in Cell Biology Biochemistry, Genetics and Molecular Biology-Cell Biology
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期刊介绍: Developed by leading scientists in the field, Current Protocols in Cell Biology is an essential reference for researchers who study the relationship between specific molecules and genes and their location, function and structure at the cellular level. Updated every three months in all formats, CPCB is constantly evolving to keep pace with the very latest discoveries and developments.
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