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A Bacterial Cell‐Free Expression System to Produce Membrane Proteins and Proteoliposomes: From cDNA to Functional Assay
Publication Name:
Current Protocols in Protein Science
Unit Number:
Unit 5.22
DOI:
10.1002/0471140864.ps0522s54
Online Posting Date:
November, 2008 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
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
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
- 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
- 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 DH5 Chemically Competent cells: F
– f80dlacZ.M15.(lacZYA-argF)U169 endA1 recA1 (Invitrogen) - SOC medium (see
- LB agar plates (appendix
- LB liquid medium (appendix
- 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
- 4× loading buffer (BioRad)
- Wide-range protein standards (see unit
- SDS-PAGE gel (Criterion XT Bis-Tris Gradient Gel, 4% to 12%, 18-well, 30-µl, BioRad; also see unit
- TBS-T (see
- Transfer buffer for semi-dry system (see
- Blocking solution for
- 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
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 (
- RTS 500 HY Kit (Roche Applied Science)
- 10 mg/ml liposome preparation (
- 50 mM Tris×Cl, pH 7.2 (appendix
- Sucrose
- Denaturing sample buffer (unit
- SDS-PAGE gel (Criterion XT Bis-Tris Gradient Gel, 4% to 12%, 18-well, 30-µl, BioRad; also see unit
- Fixative solution for silver staining (see
- 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 (unitTable 5.22.2 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--d-maltopyranoside 482.6 1.6 + + – n-Dodecyl--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 + – – aFrom http://www.roche-applied-science.com.bSymbols 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
- Homogenizer buffer (see
- Homogenate washing buffer (see
- Hypotonic buffer (see
- Discontinuous sucrose gradient: 15 ml each of 0.93 M and 0.6 M sucrose in hypotonic buffer
- Resuspension solution (see
- Chloroform
- Silica gel 60 (Merck)
- 65:25:4 (v/v/v) chloroform/methanol/H
2 O - 50:20:10:10:5 (v/v/v/v) chloroform/acetone/methanol/acetic acid/H
2 O - Nitrogen source
- 1:2 (33.3%/66.6%) chloroform/methanol
- DEPC-treated H
2 O: add 0.2 ml diethylpyrocarbonate (DEPC) per 100 ml distilled H2 O; 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
2 O [add 0.2 ml diethylpyrocarbonate (DEPC) per 100 ml distilled H2 O; 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
- MagneHis beads (Promega)
- 20 mM Tris×Cl, pH 7 to 8 (appendix
- Detergent selected in small-scale optimization tests (see
- Washing buffers 1, 2, and 3 (see
- Elution buffer (see
- SDS-PAGE gel (Criterion XT Bis-Tris Gradient Gel, 4% to 12%, 18-well, 30-µl, BioRad; also see unit
- Phosphate-buffered saline (PBS; appendix
- End-over-end rotator
- MagneSphere Magnetic Separation Stand (Promega)
- Centricon centrifugal concentrator, 10 to 30 kDa (Millipore)
- Dialysis cassettes (appendix
- Additional reagents and equipment for SDS-PAGE (unit
Basic Protocol 4: Measurement of Transduction Properties of Purified Proteoliposomes
Materials
- Cell line of interest
- Proteoliposomes containing the MP of interest (
- Phosphate-buffered saline (PBS; appendix
- Mito Tracker Red 580 (Molecular Probes)
- 4% (w/v) paraformaldehyde in PBS
- 0.1% (w/v) saponin in PBS
- Blocking solution for
- 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
- 0.5% trypsin-EDTA (e.g., Invitrogen)
- Mitochondrial buffer (see
- 2.5 M sucrose
- CFS buffer (see
- BCA Assay (Pierce; also see unit
- Proteoliposomes containing the MP of interest (
- 50 mM Tris×Cl, pH 7.4 (appendix
- Gel loading buffer (unit
- 15% denaturing SDS-PAGE gel (unit
- Tris-glycine running buffer (see
- Transfer buffer for semi-dry system (see
- 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
Basic Protocol 6: Analysis of Apoptotic Proteins by Immunoblot
Materials
- Cultured cells of interest
- Proteoliposomes containing the MP of interest (
- Phosphate-buffered saline (PBS; appendix
- 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
- Transfer buffer for semi-dry system (see
- 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
Looking for materials? Suppliers Guide
Figures
-
Figure 5.22.1Workflow for production of recombinant proteoliposomes using a classical in vivo technique and the cell-free expression system. -
Figure 5.22.2Small-scale (12- to 100-µl) expression test. The diagram depicts some of the parameters that can be optimized to enhance expression of membrane proteins. Abbreviations: GSH, reduced glutathione; GSSH, oxidized glutathione; PI, phosphoinositol; IPTG, isopropyl--d-1-thiogalactopyranoside. -
Figure 5.22.3Examples of expression of several MPs produced in small-scale reactions (25 µl). (A) VDAC-N cloned in pIVEX2.4NdeI, OEP24 cloned in pIVEX2.4NdeI, Bak cloned in pVP22-myc/HIS (Invitrogen), and GFP control. (B, C) Different levels of expression for Bak and VDAC proteins cloned into different vectors: pIVEX 2.3MCS (C-terminal His) or pIVEX 2.4NdeI (N-terminal His). -
Figure 5.22.4Purification of proteoliposomes by discontinuous sucrose gradient. Natural liposomes from thylakoid lipids are used to produce the outer envelope protein OEP24. The thin layer pointed out in the “after” illustration corresponds to the proteoliposomes fraction. The pellet is composed of insoluble proteins from the lysate and the OEP24 not integrated into the bilayer. -
Figure 5.22.5Example of gel silver staining and corresponding immunoblotting of purified proteoliposomes containing recombinant mitochondrial Bak protein. The upper panel shows silver-stained fractions from the purification of Bak proteoliposomes. The lower panel is a corresponding immunoblot of Bak proteoliposome fractions. M = Bak monomer; D = Bak dimer; T = Bak trimer. -
Figure 5.22.6Electron microscopy of Bak proteoliposomes. -
Figure 5.22.7Example of a Coomassie blue–stained gel showing purification fractions for a soluble MP. The outer envelope protein from chloroplasts, OEP24, was synthesized in the presence of detergent and then purified. E1-3, OEP24 elution fractions; B, magnetic beads (loaded to check if some protein still binds to the particles); BSA, bovine serum albumin (used as standard to estimate the amount of purified protein). -
Figure 5.22.8(A) Confocal immunolocalization of Bak proteoliposomes during a 24-hr time course. Cells treated with empty liposomes were used as negative control. (B) Cells were incubated for 6, 12, and 24 hr in the presence of Bak liposomes. Alexa Fluor 488 (green) is used as secondary antibody. MitoTracker (red) labels mitochondria and Hoechst (blue) stain the nucleus. -
Figure 5.22.9In vitro assay for cytochrome c release. Three concentrations of proteoliposomes were used: L1, L2, and L3. Pellet and supernatant were analyzed by immunoblotting using an anti-cytochrome c antibody. Control mitochondrial pellet and supernatant were loaded as well to demonstrate the integrity of the preparation. -
Figure 5.22.10Liposome structure. Liposomes are nanoparticles with various targeting ligands attached to their surface allowing for their surface attachment and accumulation in pathological areas for treatment of disease. Illustration by Kosi Gramatikoff (http://en.wikipedia.org/wiki/Image:Liposome.jpg; Torchilin,
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