G Protein–Coupled Estrogen Receptor Production Using an  Escherichia coli  Cell‐Free Expression System | Zendy

Souza Samson A. | Zendy; Kurohara Dane T. | Zendy; Dabalos Chester L. | Zendy; Ng Ho Leung | Zendy

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

Author(s) -

Souza Samson A.,

Kurohara Dane T.,

Dabalos Chester L.,

Ng Ho Leung

Publication year - 2019

Publication title -

current protocols in protein science

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 1.409

H-Index - 32

eISSN - 1934-3663

pISSN - 1934-3655

DOI - 10.1002/cpps.88

Subject(s) - escherichia coli , microbiology and biotechnology , protein expression , estrogen receptor , chemistry , receptor , biology , biochemistry , gene , genetics , cancer , breast cancer

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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