G Protein-Coupled Estrogen Receptor Production Using an Escherichia coli Cell-Free Expression System.
Curr Protoc Protein Sci
; 97(1): e88, 2019 09.
Article
em En
| MEDLINE
| ID: mdl-31517450
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.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Receptores Acoplados a Proteínas G
/
Escherichia coli
Limite:
Humans
Idioma:
En
Revista:
Curr Protoc Protein Sci
Ano de publicação:
2019
Tipo de documento:
Article