Mammalian membrane receptors expression as inclusion bodies in Escherichia coli

Methods in Molecular Biology
Bernard Mouillac, Jean-Louis Banères

Abstract

Integral membrane proteins, in particular receptors, regulate numerous physiological functions. The primary difficulty presented by their study in vitro is to obtain them in sufficient amounts in a functional state. Escherichia coli is a host of choice for producing recombinant proteins for structural studies. However, insertion of G-protein coupled receptors into its plasma membrane usually results in bacterial death. An alternative approach consists of targeting recombinant receptors to inclusion bodies, where they accumulate without affecting bacterial growth, and then fold them in vitro . We describe here a general approach that consists of accumulating the receptor in bacterial inclusion bodies, then purifying it under denaturing conditions. A simple assay is then described to screen for refolding conditions of the protein.

Citations

Jan 25, 2013·Protein Science : a Publication of the Protein Society·Pin-Chuan SuBryan W Berger
Jun 12, 2013·Langmuir : the ACS Journal of Surfaces and Colloids·Jana Broecker, Sandro Keller

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