Open AccessG 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.