Cell‐Free Expression of G Protein–Coupled Receptors

Kenneth Segers1, Stefan Masure1

1 Structural Biology Group, Biologics Research Europe, Janssen Research & Development, Beerse, Belgium
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 29.14
DOI:  10.1002/0471140864.ps2914s81
Online Posting Date:  August, 2015
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Abstract

The large‐scale production of recombinant G protein–coupled receptors (GPCRs) is one of the major bottlenecks that hamper functional and structural studies of this important class of integral membrane proteins. Heterologous overexpression of GPCRs often results in low yields of active protein, usually due to a combination of several factors, such as low expression levels, protein insolubility, host cell toxicity, and the need to use harsh and often denaturing detergents (e.g., SDS, LDAO, OG, and DDM, among others) to extract the recombinant receptor from the host cell membrane. Many of these problematic issues are inherently linked to cell‐based expression systems and can therefore be circumvented by the use of cell‐free systems. In this unit, we provide a range of protocols for the production of GPCRs in a cell‐free expression system. Using this system, we typically obtain GPCR expression levels of ∼1 mg per ml of reaction mixture in the continuous‐exchange configuration. Although the protocols in this unit have been optimized for the cell‐free expression of GPCRs, they should provide a good starting point for the production of other classes of membrane proteins, such as ion channels, aquaporins, carrier proteins, membrane‐bound enzymes, and even large molecular complexes. © 2015 by John Wiley & Sons, Inc.

Keywords: cell‐free expression; E. coli extract; membrane protein; GPCR; detergent; lipid

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Extract Testing and Optimization of Cell‐Free Reaction Conditions
  • Support Protocol 1: Preparation of E. coli S12 Extract
  • Support Protocol 2: Preparation of E. coli S12 T7 RNA Polymerase Extract
  • Basic Protocol 2: Design and Preparation of DNA Templates for Cell‐Free Membrane Protein Expression
  • Basic Protocol 3: Cell‐Free Expression of Membrane Proteins as Precipitates in Batch Configuration
  • Basic Protocol 4: Cell‐Free Expression of Membrane Proteins as Precipitates in Continuous‐Exchange Configuration
  • Basic Protocol 5: Cell‐Free Production of Soluble Membrane Proteins
  • Alternate Protocol 1: Cell‐Free Expression of Membrane Proteins in the Presence of Detergents (SD‐CF) or the Amphipatic Polymer NVoy (SN‐CF)
  • Alternate Protocol 2: Cell‐Free Expression of Membrane Proteins in the Presence of Lipids (L‐CF)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Extract Testing and Optimization of Cell‐Free Reaction Conditions

  Materials
  • E. coli S12 cell extract (see protocol 2)
  • E. coli S12‐T7 cell extract (see protocol 3)
  • 100 mg/ml NaN 3 (Sigma)
  • 400 mg/ml PEG8000 (Sigma)
  • 4 M potassium acetate (KOAc; Sigma)
  • 2.4 M HEPES (Sigma; adjust to pH 8.0 with KOH)
  • 50× Complete protease inhibitor cocktail without EDTA (Roche Diagnostics); prepare by dissolving ten tablets in 10 ml of Milli‐Q water
  • 10 mg/ml folinic acid calcium salt (Sigma)
  • NTP mix (33.3 mM ATP, 22.2 mM UTP, 22.2 mM GTP, 22.2 mM CTP; see recipe)
  • 430 mM phospo(enol)pyruvic acid monopotassium salt (PEP; Sigma; adjust to pH 7.0 with KOH)
  • 1 M acetyl phosphate lithium potassium salt (AcP; Sigma; adjust to pH 7.0 with KOH)
  • Amino acid mix (4 mM each; see recipe)
  • RCWMDE amino acid mix (16.7 mM each, see recipe)
  • 1 M magnesium acetate tetrahydrate [Mg(OAc) 2; Sigma]
  • 10 mg/ml pyruvate kinase (Roche Diagnostics)
  • 40 U/μl Ribolock RNase (ribonuclease) inhibitor (Thermo Scientific)
  • 40 mg/ml E. coli total transfer RNA (tRNA; Roche Diagnostics)
  • 2.8 M potassium acetate (KOAc; anhydrous; Sigma)
  • 200 ng/μl plasmid DNA encoding GFP or eGFP (standard E. coli expression vectors like pIVEX or pET derivatives can be used)
  • Rolling device or temperature‐controlled shaking incubator
  • Black Corning Costar 384‐well plate
  • Fluorescence 96‐/384‐well plate reader
NOTE: The cell‐free transcription/translation procedure is quite sensitive to the presence of RNases. Therefore, gloves should be worn at all times while making up the solutions and setting up the reactions. Disposable sterile plasticware or autoclaved glassware should be used throughout. All chemicals are purchased as analytical‐quality, RNase‐free grade. Prepare all solutions with RNase‐free Milli‐Q water and keep at −20°C if not stated otherwise. Store all solutions in small working aliquots at −20°C to avoid repeated freeze‐thaw cycles.

Support Protocol 1: Preparation of E. coli S12 Extract

  Materials
  • Superior Broth (Athena Enzyme Systems)
  • E. coli strain BL21 Star (DE3) glycerol stock (Life Technologies)
  • S12 extract buffer A (see recipe)
  • S12 extract buffer B (see recipe)
  • Ultra Yield Flask, 2.5 liter (Thomson)
  • Autoclave indicator tape
  • Ultra Yield Flask Air‐Porous Seal (Thomson)
  • Temperature‐controlled shaking incubator
  • Spectrophotometer (capable of measuring absorbance at 600 nm)
  • Refrigerated centrifuge
  • French pressure cell or high‐pressure cell homogenizer

Support Protocol 2: Preparation of E. coli S12 T7 RNA Polymerase Extract

  Additional Materials (also see protocol 2)
  • Isopropyl‐β‐D‐thiogalactopyranoside (IPTG)

Basic Protocol 2: Design and Preparation of DNA Templates for Cell‐Free Membrane Protein Expression

  Materials
  • Cell‐free expression plasmid (e.g., pIVEX2.4 d)
  • DNA encoding the gene of interest (e.g., a genomic or cDNA library)
  • Additional reagents and equipment for the polymerase chain reaction (PCR; Kramer and Coen, ), DNA sequencing (Shendure et al., ), and general molecular cloning techniques (Ausubel et al., )

Basic Protocol 3: Cell‐Free Expression of Membrane Proteins as Precipitates in Batch Configuration

  Materials
  • E. coli S12 cell extract (see protocol 2)
  • E. coli S12‐T7 cell extract (see protocol 3)
  • 100 mg/ml NaN 3 (Sigma)
  • 400 mg/ml PEG8000 (Sigma)
  • 4 M potassium acetate (KOAc; Sigma)
  • 2.4 M HEPES (Sigma; adjust to pH 8.0 with KOH)
  • 50× Complete protease inhibitor cocktail without EDTA (Roche Diagnostics); prepare by dissolving ten tablets in 10 ml of Milli‐Q water
  • 10 mg/ml folinic acid calcium salt (Sigma)
  • NTP mix (33.3 mM ATP, 22.2 mM UTP, 22.2 mM GTP, 22.2 mM CTP; see recipe)
  • 430 mM phospo(enol)pyruvic acid monopotassium salt (PEP; Sigma); adjust to pH 7.0 with KOH)
  • 1 M acetyl phosphate lithium potassium salt (AcP; Sigma; adjust to pH 7.0 with KOH)
  • Amino acid mix (4 mM each; see recipe)
  • RCWMDE amino acid mix (16.7 mM each, see recipe)
  • 10 M magnesium acetate tetrahydrate (Mg(OAc) 2; Sigma)
  • 10 mg/ml pyruvate kinase (Roche Diagnostics)
  • 40/μl Ribolock RNase (ribonuclease) inhibitor (Thermo Scientific)
  • 40 mg/ml E. coli total transfer RNA (tRNA; Roche Diagnostics)
  • 2.8 M potassium acetate (KOAc; anhydrous; Sigma)
  • 200 ng/μl plasmid DNA encoding the membrane protein target
  • Solubilization buffer: 20 mM Tris·Cl, pH 7.5/150 mM NaCl containing 1× Complete protease inhibitor cocktail without EDTA (Roche Diagnostics)
  • Rolling device or temperature‐controlled shaking incubator
  • Refrigerated microcentrifuge
  • Additional reagents and equipment for SDS‐PAGE (Gallagher, ) and western blotting (immunoblotting; Gallagher et al., )

Basic Protocol 4: Cell‐Free Expression of Membrane Proteins as Precipitates in Continuous‐Exchange Configuration

  Materials
  • E. coli S12 cell extract (see protocol 2)
  • E. coli S12‐T7 cell extract (see protocol 3)
  • 100 mg/ml NaN 3 (Sigma)
  • 400 mg/ml PEG8000 (Sigma)
  • 4 M potassium acetate (KOAc; Sigma)
  • 2.4 M HEPES (Sigma; adjust to pH 8.0 with KOH)
  • 50× Complete protease inhibitor cocktail without EDTA (Roche Diagnostics); prepare by dissolving ten tablets in 10 ml of Milli‐Q water
  • 10 mg/ml folinic acid calcium salt (Sigma)
  • NTP mix (33.3 mM ATP, 22.2 mM UTP, 22.2 mM GTP, 22.2 mM CTP; see recipe)
  • 430 mM phospo(enol)pyruvic acid monopotassium salt (PEP; Sigma; adjust to pH 7.0 with KOH)
  • 1 M acetyl phosphate lithium potassium salt (AcP; Sigma; adjust to pH 7.0 with KOH)
  • Amino acid mix (4 mM each; see recipe)
  • RCWMDE amino acid mix (16.7 mM each, see recipe)
  • 1 M magnesium acetate tetrahydrate [Mg(OAc) 2; Sigma]
  • 10 mg/ml pyruvate kinase (Roche Diagnostics)
  • 40/μl Ribolock RNase (ribonuclease) inhibitor (Thermo Scientific)
  • 40 mg/ml E. coli total transfer RNA (tRNA; Roche Diagnostics)
  • 2.8 M potassium acetate (KOAc; anhydrous; Sigma)
  • 200 ng/μl plasmid DNA encoding the membrane protein target
  • Solubilization buffer: 20 mM Tris·Cl, pH 7.5 ( appendix 2E)/150 mM NaCl containing 1× Complete protease inhibitor cocktail without EDTA (Roche Diagnostics)
  • Plastic vials, Ø 15 mm (GE Healthcare, BR‐1006‐54)
  • D‐Tube Dialyzer Mini (MWCO 12 to 14 kDa; Novagen)
  • Rubber caps, type 5 (GE Healthcare, BR‐1006‐55)
  • Rolling device or temperature‐controlled shaking incubator
  • Additional reagents and equipment for SDS‐PAGE (Gallagher, ) and western blotting (immunoblotting; Gallagher et al., )

Basic Protocol 5: Cell‐Free Production of Soluble Membrane Proteins

  Materials
  • P‐CF expressed membrane protein (see Basic Protocols protocol 53 and protocol 64) in solubilization buffer
  • Resuspension buffer: 20 mM Tris·Cl, pH 7.5 ( appendix 2E)/150 mM NaCl containing 1× Complete protease inhibitor cocktail without EDTA and 1 mM DTT
  • Solubilization buffer: same as resuspension buffer, but supplemented with an appropriate detergent (high‐purity grade); detergent concentration should be higher than the specific critical micellar concentration (CMC), e.g., 3‐ to 5‐fold higher than their respective CMC.
  • Refrigerated microcentrifuge
  • Rolling device or shaking incubator
  • Additional reagents and equipment for SDS‐PAGE (Gallagher, ) and western blotting (immunoblotting; Gallagher et al., )

Alternate Protocol 1: Cell‐Free Expression of Membrane Proteins in the Presence of Detergents (SD‐CF) or the Amphipatic Polymer NVoy (SN‐CF)

  Additional Materials (also see Basic Protocols protocol 53 and protocol 64)
  • Detergents: stock solutions of the selected detergents (high‐purity grade) in Milli‐Q water; the stock concentration should be at least 10 times higher than the final working concentration (see Table 29.14.6).
  • NVoy polymer (Expedeon)
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Literature Cited

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