G Protein-Coupled Estrogen Receptor Production Using an Escherichia coli Cell-Free Expression System.
G Protein-Coupled Estrogen Receptor Production Using an Escherichia coli Cell-Free Expression System.
Curr Protoc Protein Sci. 2019 Sep;97(1):e88
Authors: Souza SA, Kurohara DT, Dabalos CL, Ng HL
Abstract
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.
PMID: 31517450 [PubMed - in process]
Source: Current Protocols in Protein Science - Category: Biochemistry Tags: Curr Protoc Protein Sci Source Type: research