G Protein-Coupled Receptors

Gabriel Berstein

• Publisher: CRC Press
• Year: 2019
• eBook ISBN: 9780429525261
• Topic: Médecine
• Subtopic: Biochimie en médecine

Covering recently developed methods in membrane-bound receptors, this book emphasizes receptor structure and function, knowledge of which is essential to the study of signal transduction. G Protein-Coupled Receptors has culled contributors from domestic and international sources, providing a broad base of knowledge. Some topics covered are the r

Section III

3D Structure: Large-Scale Expression and Electron- and X-Ray Crystallography

10	Expression in Escherichia coli and Large-Scale Purification of a Rat Neurotensin Receptor Reinhard Grisshammer and Julie Tucker

CONTENTS

10.1 Introduction

10.2 Expression of NTR in Escherichia Coli

10.2.1 Background

10.2.2 Methods for Receptor Expression

10.3 Purification of the Fusion Protein MBP-T43NTR-TrxA-H10

10.3.1 General Considerations for Solubilization and Purification

10.3.2 Methods for Receptor Purification

10.3.2.1 Cell Lysis and Solubilization

10.3.2.2 Immobilized Metal Affinity Chromatography (IMAC)

10.3.2.3 Neurotensin Column

10.4 Ligand Binding Analysis of Solubilized NTR

10.5 Determination of Protein Content

10.6 Conclusions

Acknowledgments

References

10.1 INTRODUCTION

Seven-helix G protein-coupled receptors (GPCRs) are integral components of the cell surface membrane.^1,2,3 Estimates suggest that there may be several thousand GPCRs in the human genome, of which a substantial proportion could be drug targets. Therefore, it is important to understand the molecular structure and action of GPCRs. Only rhodopsin, the naturally abundant visual pigment, has been crystallized to date. Projection structures at low resolution^4,5,6,7 and the three-dimensional arrangement of the transmembrane alpha helices^8,9 have been determined by electron cryomicroscopy using two-dimensional crystals.

In contrast to rhodopsin, most GPCRs do not occur naturally in large quantities. Structural work therefore requires an amenable expression system that permits routine purification of milligram quantities of protein. The potential of various hosts for the expression of membrane proteins has recently been discussed in detail^10,11 (see also Chapter 11). Our work focuses on the high-affinity receptor for neurotensin from rat (NTR)¹² using Escherichia coli as the expression system. We describe here the expression of functional NTR inserted in E. coli membranes, but do not discuss the production of NTR in nonfunctional, aggregated form.^12a

For crystallization, purified receptors should be fully functional and homogeneous; thus the purification procedure has to be fast and efficient, as receptors often show reduced stability in the detergents used to extract them from membranes. In this chapter we discuss a two-step purification scheme to obtain functional NTR based on immobilized metal affinity chromatography (IMAC) followed by an agonist ligand column step. We have not yet investigated the refolding of NTR from inclusion bodies, which could also be a route to obtain functional receptors in quantities sufficient for biochemical and structural analyses.¹³

10.2 EXPRESSION OF NTR IN ESCHERICHIA COLI

10.2.1 BACKGROUND

The great attraction of E. coli as an expression host is the simplicity of its use. The time required for the construction of recombinant expression vectors and the initial expression experiment is very short in comparison to other host systems. This allows rapid optimization of expression yields and testing of affinity purification tags.¹⁴ Low-cost, large-scale fermentation and homogeneous protein expression (few post-translational modifications) may be advantageous for biophysical and structural studies.

E. coli has been used successfully for the expression of GPCRs in functional, membrane-inserted form (see Table 10.1), despite being very different from eukary-otic expression systems.¹⁵ N-linked glycosylation and cholesterol (a major constituent of mammalian plasma membranes but absent from E. coli membranes) are often not required for expression of functional receptors. A brief summary of parameters important for the expression of NTR in E. coli is given below. The reader is referred to Reference 15 for a more detailed discussion of the folding and membrane insertion of GPCRs related to their expression in E. coli.

Expression of the receptor-encoding cDNA alone usually results in very low levels of protein. In contrast, higher expression levels can be achieved by using a fusion protein approach, in which the E. coli periplasmic maltose-binding protein (MBP) with its signal peptide is linked to the amino terminus of the receptor. This may facilitate the correct orientation of the receptor in the cytoplasmic membrane with its N terminus in the periplasmic space. The presence of MBP does not seem to interfere with the ligand binding properties of recombinant receptors.^{16,17,18,19,20} We found that the pharmacology of NTR expressed in E. coli is similar to that of NTR expressed in eukaryotic cells and from native tissues.^14,18,18a

TABLE 10.1
Expression of GPCRs in E. coli

Abbreviations: aa, amino acids; C, receptor carboxy terminus; CGT, cyclomaltodextrin glucanotransferase; GST, glutathione S-transferase; ib, inclusion body; m, membrane; MBP, maltose-binding protein; N, receptor amino terminus; n.a., not available; SP, signal peptide; TrxA, E. coli thioredoxin.

^a Expression levels are given in pmol/mg of membrane protein and receptors/cell (ligand binding analysis) or mg of receptor/liter of culture.

^b Induction conditions are given as medium, time of induction, and temperature; n.a., not available.

^c CB1 receptor is poorly expressed due to extensive proteolysis (Western blot analysis) and fails to bind ligand.

^d Endothelin ET_B receptor has its own putative signal peptide, whereas the other receptors listed in this table do not.

Adapted from Reference 15.

No GPCR has yet been overexpressed in E. coli in functional form at levels above 100 pmol/mg of membrane protein. The process of membrane insertion and folding into the native receptor conformation seems to be rate-limiting. To avoid overloading of the E. coli insertion/translocation machinery, transcription and translation were optimized rather than maximized by using a low copy number plasmid with a weak promoter for expression of the NTR fusion protein (see below). Furthermore, low temperature (20°C) and prolonged induction times (>40 h) gave greater expression. Quantitative data are not yet available on the relative proportions of correctly folded and misfolded receptors in the cytoplasmic membrane of E. coli.

The stability of GPCRs can be an important factor for their successful expression, as was found for NTR.¹⁴ Whe...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Table of Contents
• Section I Novel Ligands: Ligand Screening Systems and Orphan Receptors
• Section II Function and Regulation: Receptor-G Protein Coupling and Desensitization
• Section III 3D Structure: Large-Scale Expression and Electron- and X-Ray Crystallography