Expression and Purification of Functional Human Mu Opioid Receptor from E.coli

Yanbin Ma,Jörg Labahn,Jan Kubicek,Jian R Lu

doi:10.1371/journal.pone.0056500

Abstract

N-terminally his-tagged human mu opioid receptor, a G protein-coupled receptor was produced in E.coli employing synthetic codon-usage optimized constructs. The receptor was expressed in inclusion bodies and membrane-inserted in different E.coli strains. By optimizing the expression conditions the expression level for the membrane-integrated receptor was raised to 0.3–0.5 mg per liter of culture. Milligram quantities of receptor could be enriched by affinity chromatography from IPTG induced cultures grown at 18°C. By size exclusion chromatography the protein fraction with the fraction of alpha-helical secondary structure expected for a 7-TM receptor was isolated, by CD-spectroscopy an alpha-helical content of ca. 45% was found for protein solubilised in the detergent Fos-12. Receptor in Fos-12 micelles was shown to bind endomorphin-1 with a KD of 61 nM. A final yield of 0.17 mg functional protein per liter of culture was obtained.

Highlights

G protein-coupled receptors (GPCRs) are the largest family of integral membrane proteins which account for up to 50% of all drug targets including cardiovascular and gastrointestinal diseases, central nervous system and immune disorders, cancer and pain [1,2,3,4,5].Opioid receptors have been classified into three different types, m, d, k [6]
The m type human mu-opioid receptor OPRM is activated by endogenous opioid peptides such as beta-endorphins and exogenous alkaloids such as morphine
At high temperature (37uC), the N-terminal his-tagged OPRM was found to be produced both in inclusion bodies and in membrane-inserted form (Figure 1A): for C41 cells only a low expression level was observed, most of the target protein was found in the inclusion bodies

Summary

Introduction

G protein-coupled receptors (GPCRs) are the largest family of integral membrane proteins which account for up to 50% of all drug targets including cardiovascular and gastrointestinal diseases, central nervous system and immune disorders, cancer and pain [1,2,3,4,5].Opioid receptors have been classified into three different types, m, d, k [6]. G protein-coupled receptors (GPCRs) are the largest family of integral membrane proteins which account for up to 50% of all drug targets including cardiovascular and gastrointestinal diseases, central nervous system and immune disorders, cancer and pain [1,2,3,4,5]. The m type human mu-opioid receptor OPRM is activated by endogenous opioid peptides such as beta-endorphins and exogenous alkaloids such as morphine. GPCRs represents major pharmaceutical targets, only few structural data on GPCRs have been obtained. This is mainly due to the hydrophobicity of these proteins, very low natural abundance, difficulties in overexpression and purification and low stability after extraction from the membrane environment [9]. The crystal structure of human OPRM with T4 lysozyme inserted in 3rd intracellular loop was determined [10]

Methods

Results

Conclusion