利用大肠杆菌无细胞表达系统制备G蛋白偶联雌激素受体

Q1 Biochemistry, Genetics and Molecular Biology Current Protocols in Protein Science Pub Date : 2019-06-19 DOI:10.1002/cpps.88

Samson A. Souza, Dane T. Kurohara, Chester L. Dabalos, Ho Leung Ng

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引用次数: 2

摘要

G蛋白偶联雌激素受体(GPER)的异源表达伴随着膜蛋白固有的一系列挑战。该受体在大肠杆菌和酵母中的低表达水平和倾向于形成不溶性聚集体使其成为一个难以研究的受体靶点。在本单元中，我们详细介绍了使用基于沉淀的无细胞系统生产单体GPER的步骤。我们提供了用于表达的DNA结构的信息，用于生成主混合物的反应补充的移液方案，以及无细胞反应设置。在本单元的最后一部分，我们概述了增溶和纯化的步骤，并提供了一种通过液相色谱-质谱检测定性观察功能的可行方法。©2019 by John Wiley &儿子,Inc。

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G Protein–Coupled Estrogen Receptor Production Using an Escherichia coli Cell-Free Expression System

Heterologous expression of the G protein–coupled estrogen receptor (GPER) comes with a suite of challenges intrinsic to membrane proteins. This receptor's low expression levels and tendency to form insoluble aggregates in Escherichia coli and yeast make it a difficult receptor-target to study. In this unit, we detail steps to produce monomeric GPER using a precipitation-based cell-free system. We provide information on the DNA construct for expression, the pipetting scheme for the reaction supplements to generate a master mix, and the cell-free reaction setup. In the last portion of this unit, we outline steps for solubilization and purification, and we provide a viable method for qualitatively observing functionality by liquid chromatography–mass spectrometry detection. © 2019 by John Wiley & Sons, Inc.

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Current Protocols in Protein Science Biochemistry, Genetics and Molecular Biology-Biochemistry

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期刊介绍： With the mapping of the human genome, more and more researchers are exploring protein structures and functions in living organisms. Current Protocols in Protein Science provides protein scientists, biochemists, molecular biologists, geneticists, and others with the first comprehensive suite of protocols for this growing field.

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