A Bacterial Cell‐Free Expression System to Produce Membrane Proteins and Proteoliposomes: From cDNA to Functional Assay

Lavinia Liguori1, Bruno Marques1, Jean‐Luc Lenormand1

1 HumProTher Laboratory, TheREx‐GREPI, TIMC‐IMAG Laboratory, University of Joseph Fourier, UFR de Médecine, La Tronche, France
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.22
DOI:  10.1002/0471140864.ps0522s54
Online Posting Date:  November, 2008
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Abstract

Limitations in the production of folded membrane proteins represent the major bottleneck for functional and structural studies of this huge category of macromolecules. Cell‐free expression systems provide an attractive alternative to the classical overexpression systems for producing membrane proteins. However, optimization of these systems remains a challenging task, considering the hydrophobic properties of these molecules. This unit describes the production of eukaryotic membrane proteins either in soluble form or integrated into liposomes using a bacterial cell‐free expression system. Liposomes in the reaction mixture induce the direct insertion of freshly produced membrane proteins into the bilayer and allow the formation of functional proteoliposomes in which the membrane proteins are correctly folded. Curr. Protoc. Protein Sci. 54:5.22.1‐5.22.30. © 2008 by John Wiley & Sons, Inc.

Keywords: cell‐free expression system; membrane proteins production; one‐step proteoliposomes synthesis; delivery system

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Cloning and Small‐Scale (25‐ to 100‐µl) Expression of Membrane Proteins Using a Bacterial Cell‐Free System
  • Basic Protocol 2: Production and Purification of Proteoliposomes and Soluble Membrane Proteins
  • Support Protocol 1: Preparation of Liposomes from Spinach Thylakoid Membrane Lipids
  • Support Protocol 2: Preparation of Liposomes from Synthetic Lipids
  • Basic Protocol 3: Cell‐Free Production of Soluble His‐Tagged Membrane Proteins in Detergent
  • Basic Protocol 4: Measurement of Transduction Properties of Purified Proteoliposomes
  • Basic Protocol 5: Functionality Tests for Recombinant Proteoliposomes: In Vitro Assay for Mitochondrial Cytochrome c Release
  • Basic Protocol 6: Analysis of Apoptotic Proteins by Immunoblot
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Cloning and Small‐Scale (25‐ to 100‐µl) Expression of Membrane Proteins Using a Bacterial Cell‐Free System

  Materials
  • 10× PCR buffer ( appendix 4J)
  • 10 mM dNTP Mix (Roche Applied Science)
  • Template DNA: cDNA sequences of interest for expression
  • 25 mM MgCl 2
  • Proofreading DNA polymerase: e.g., PfuUltra High‐Fidelity DNA Polymerase (Stratagene) or Isis Proofreading DNA Polymerase (QBiogene)
  • 1% agarose gel in TAE buffer with ethidum bromide ( appendix 4F)
  • QIAquick Gel Extraction kit (Qiagen)
  • pIVEX2.3d or pIVEX2.4d plasmids (Roche Applied Science)
  • Restriction enzymes (e.g., NdeI/XhoI; Roche Applied Science)
  • Rapid DNA Ligation Kit (with T4 DNA ligase; Roche Applied Science)
  • Library Efficiency DH5α Chemically Competent cells: F f80dlacZ.M15.(lacZYA‐argF)U169 endA1 recA1 (Invitrogen)
  • SOC medium (see recipe)
  • LB agar plates ( appendix 4A) containing 100 µg/ml ampicillin (10‐cm diameter)
  • LB liquid medium ( appendix 4A) containing 100 µg/ml ampicillin
  • QIAprep Spin Miniprep Kit (Qiagen)
  • HiSpeed Plasmid Midi Kit (Qiagen)
  • Rapid Translation System (RTS) 100 HY Kit (Roche Applied Science)
  • 100× GSH‐GSSG: 10 mM GSH:100 mM GSSG (both reagents available from Sigma‐Aldrich; optional)
  • GroE (Roche Applied Science; optional)
  • DnaK (Roche Applied Science; optional)
  • Complete Protease Inhibitor cocktail tablets, Mini, EDTA‐free (Roche Applied Science; optional)
  • Pefabloc SC (Roche Applied Science)
  • Leupeptin (Sigma‐Aldrich; optional)
  • Pepstatin (Sigma‐Aldrich; optional)
  • NV10 polymers (Novexin; optional)
  • 50 mM Tris⋅Cl, pH 7.5 ( appendix 2E)
  • 4× loading buffer (BioRad)
  • Wide‐range protein standards (see unit 10.1)
  • SDS‐PAGE gel (Criterion XT Bis‐Tris Gradient Gel, 4% to 12%, 18‐well, 30‐µl, BioRad; also see unit 10.1) in MES buffer
  • TBS‐T (see recipe)
  • Transfer buffer for semi‐dry system (see recipe)
  • Blocking solution for protocol 1 (see recipe)
  • Monoclonal anti‐His HRP‐conjugated antibody (Sigma‐Aldrich, cat. no. A7058)
  • ECL Western Blotting Detection Kit (GE Healthcare)
  • BioRad iCycler Thermal Cycler (BioRad)
  • 42°C water bath
  • 14‐cm tubes
  • Shaking platform
  • RTS ProteoMaster (Roche Applied Sciences)
  • 0.22‐ or 0.45‐µm pore size nitrocellulose membrane (BioRad)
  • Hyperfilm ECL (18 × 24 cm; GE Healthcare)
  • Additional reagents and equipment for PCR ( appendix 4J), agarose gel electrophoresis ( appendix 4F), SDS‐PAGE (unit 10.1), electroblotting (unit 10.7), and Ponceau S staining of blots (unit 10.8)
CAUTION: Ethidium bromide is harmful if swallowed and is very toxic if inhaled. It is irritating to the eyes, respiratory system and skin.

Basic Protocol 2: Production and Purification of Proteoliposomes and Soluble Membrane Proteins

  Materials
  • Expression vector containing the cDNA encoding the protein of interest ( protocol 1)
  • RTS 500 HY Kit (Roche Applied Science)
  • 10 mg/ml liposome preparation ( protocol 3 or protocol 42)
  • 50 mM Tris⋅Cl, pH 7.2 ( appendix 2E)
  • Sucrose
  • Denaturing sample buffer (unit 10.1)
  • SDS‐PAGE gel (Criterion XT Bis‐Tris Gradient Gel, 4% to 12%, 18‐well, 30‐µl, BioRad; also see unit 10.1) in MES buffer
  • Fixative solution for silver staining (see recipe)
  • Silver staining solutions A: 2% (w/v) silver nitrate/2% (w/v) ammonium nitrate
  • Silver staining solution B: 10% (w/v) tungstosilic acid
  • Silver staining solution C: 3% (v/v) formaldehyde
  • Developer solution: 5% (w/v) sodium carbonate
  • Stop solution: 5% (v/v) acetic acid
  • Coomassie blue staining solution (Euromedex; http://www.euromedex.com)
  • RTS ProteoMaster (Roche Applied Science)
  • 0.2‐µm filters
  • 10‐ml ultracentrifuge tubes and stable tube rack
  • Ultracentrifuge
  • Boiling water bath
  • Transmission electron microscope (optional): e.g., Philips CM12 electron microscope equipped with a LaB6 filament operating at 100 kV.
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1), imaging of gels (unit 10.12), and transmission electron microscopy (optional; unit 17.2)
    Table 5.2.2   Materials   Detergents Compatible with the Rapid Translation System (Roche) ba , cb   Detergents Compatible with the Rapid Translation System (Roche)

    Detergent Molecular weight (Da) CMC (mM) <CMC =CMC >CMC
    Ionic
    Deoxycholic acid 414.6 2‐6 +
    Nonionic
    APO‐10 218.3 4.6 +
    Brij 35 1199.6 0.09 + +
    Brij 58P 1122 0.077 + + +
    Decyl‐β‐D‐maltopyranoside 482.6 1.6 + +
    n‐Dodecyl‐β‐D‐maltoside 510.6 0.1‐0.6 nd +
    Mega‐8 321.5 58 +
    Mega‐10 349.5 6‐7 + +
    NP‐40 603.0 0.05‐0.3 + + +
    Triton X‐100 625 0.2‐0.9 + + +
    Triton X‐114 558.75 0.35 + +
    Tween 20 1228 0.06 + +
    Zwitterionic
    CHAPS 614.9 6‐10 + +
    Zwittergent 3‐12 335.6 2‐4 +
    Zwittergent 3‐14 363.6 0.1‐0.4 +

     bFrom http://www.roche‐applied‐science.com.
     cSymbols and abbreviations: +, suitable for use in RTS E. coli reactions; −, expression yield in RTS 500 E. coli HY decreases more than 20%; nd, not determined; CMC, critical micellar concentration.

Support Protocol 1: Preparation of Liposomes from Spinach Thylakoid Membrane Lipids

  Materials
  • 2 kg spinach leaves (Spinacia oleracea L.)
  • Leaf washing buffer (see recipe)
  • Homogenizer buffer (see recipe)
  • Homogenate washing buffer (see recipe)
  • Hypotonic buffer (see recipe)
  • Discontinuous sucrose gradient: 15 ml each of 0.93 M and 0.6 M sucrose in hypotonic buffer
  • Resuspension solution (see recipe)
  • Chloroform
  • Silica gel 60 (Merck)
  • 65:25:4 (v/v/v) chloroform/methanol/H 2O
  • 50:20:10:10:5 (v/v/v/v) chloroform/acetone/methanol/acetic acid/H 2O
  • Nitrogen source
  • 1:2 (33.3%/66.6%) chloroform/methanol
  • DEPC‐treated H 2O: add 0.2 ml diethylpyrocarbonate (DEPC) per 100 ml distilled H 2O; shake vigorously to dissolve DEPC; autoclave the solution to inactivate remaining DEPC
  • Waring Blendor (4‐liter capacity)
  • 50‐µm gauze filters (CellTrics, Partec)
  • Sorvall RC5 centrifuge with GS‐3, HS‐4, and SS‐34 rotors
  • Beckman L2 65B ultracentrifuge with SW 28 rotor
  • SpeedVac evaporator
  • Glass chromatography column (15‐ml capacity, 1‐cm diameter)
  • Glass wool
  • Probe sonicator (e.g., Branson Sonic Power)
  • 0.22‐µm filter

Support Protocol 2: Preparation of Liposomes from Synthetic Lipids

  Materials
  • Lipids (e.g., Avanti Polar Lipids)
  • 1:1 (v/v) methanol/chloroform
  • HEPES buffer, pH 7.4 or DEPC‐treated H 2O [add 0.2 ml diethylpyrocarbonate (DEPC) per 100 ml distilled H 2O; shake vigorously to dissolve DEPC; autoclave the solution to inactivate remaining DEPC]
  • Probe sonicator (e.g., Branson Sonic Power)
  • 0.22‐µm filter

Basic Protocol 3: Cell‐Free Production of Soluble His‐Tagged Membrane Proteins in Detergent

  Materials
  • Optimized expression reaction mixture (from protocol 1)
  • MagneHis beads (Promega)
  • 20 mM Tris⋅Cl, pH 7 to 8 ( appendix 2E) or HEPES buffer, pH 7 to 8
  • Detergent selected in small‐scale optimization tests (see protocol 1)
  • Washing buffers 1, 2, and 3 (see reciperecipes)
  • Elution buffer (see recipe)
  • SDS‐PAGE gel (Criterion XT Bis‐Tris Gradient Gel, 4% to 12%, 18‐well, 30‐µl, BioRad; also see unit 10.1) in MES buffer
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • End‐over‐end rotator
  • MagneSphere Magnetic Separation Stand (Promega)
  • Centricon centrifugal concentrator, 10 to 30 kDa (Millipore)
  • Dialysis cassettes ( appendix 3B)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1), Coomassie blue staining of gels (see protocol 2), and dialysis ( appendix 3B)

Basic Protocol 4: Measurement of Transduction Properties of Purified Proteoliposomes

  Materials
  • Cell line of interest
  • Proteoliposomes containing the MP of interest ( protocol 2)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • Mito Tracker Red 580 (Molecular Probes)
  • 4% (w/v) paraformaldehyde in PBS
  • 0.1% (w/v) saponin in PBS
  • Blocking solution for protocol 6 (see recipe)
  • Primary antibody (anti‐mouse or anti‐rabbit against the MP of interest)
  • Secondary antibody (Alexa Fluor 488–conjugated anti‐mouse or anti‐rabbit; Molecular Probes)
  • Counterstain: DAPI or Hoechst 33258 (Molecular Probes)
  • Mounting medium for fluorescence procedures (Sigma, cat. no. 1000‐4)
  • Four‐well chamber slides (Nalgene Nunc International)
  • 37°C, 5% CO 2 incubator
  • Epifluorescence microscope (e.g., inverted Nikon Eclipse TE2000‐E equipped with epifilters for the different fluorochromes) or confocal microscope (e.g., Leica confocal laser scanning microscope TCS‐SP2 operating system)

Basic Protocol 5: Functionality Tests for Recombinant Proteoliposomes: In Vitro Assay for Mitochondrial Cytochrome c Release

  Materials
  • Cultured cells for mitochondria isolation
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • 0.5% trypsin‐EDTA (e.g., Invitrogen)
  • Mitochondrial buffer (see recipe)
  • 2.5 M sucrose
  • CFS buffer (see recipe)
  • BCA Assay (Pierce; also see unit 3.4)
  • Proteoliposomes containing the MP of interest ( protocol 2)
  • 50 mM Tris⋅Cl, pH 7.4 ( appendix 2E)
  • Gel loading buffer (unit 10.1)
  • 15% denaturing SDS‐PAGE gel (unit 10.1)
  • Tris‐glycine running buffer (see recipe)
  • Transfer buffer for semi‐dry system (see recipe)
  • Monoclonal anti‐cytochrome c antibody (BD Pharmingen)
  • Refrigerated centrifuge
  • Potter‐Elvehjem homogenizer
  • Additional reagents and equipment for assessing cell viability by trypan blue staining ( appendix 3C), colorimetric protein assay (unit 3.4), SDS‐PAGE (unit 10.1), transfer of protein from gel to membrane (unit 10.7), and immunoblotting (unit 10.8)

Basic Protocol 6: Analysis of Apoptotic Proteins by Immunoblot

  Materials
  • Cultured cells of interest
  • Proteoliposomes containing the MP of interest ( protocol 2)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • FastBreak Cell Lysis reagent (Promega)
  • Protease inhibitor cocktail: Complete EDTA‐Free Tabs (Roche)
  • SDS‐PAGE gel (Criterion XT Bis‐Tris Gradient Gel, 4% to 12%, 18‐well, 30‐µl, BioRad; also see unit 10.1) in MES buffer
  • Transfer buffer for semi‐dry system (see recipe)
  • Antibodies against apoptotic marker proteins, e.g.:
    • Anti‐caspase 9 antibody (Calbiochem)
    • Anti‐caspase 7 antibody (Cell Signaling Technology)
    • Anti‐PARP antibody (Cell Signaling Technology)
  • 6‐well culture plates
  • Refrigerated centrifuge
  • Additional reagents and equipment for colorimetric protein assay (unit 3.4), SDS‐PAGE (unit 10.1), transfer of proteins from gel to membrane (unit 10.7), and immunoblotting (unit 10.8)
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Figures

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Literature Cited

Literature Cited
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