High‐Throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

Renato Bruni1, Brian Kloss1

1 New York Consortium on Membrane Protein Structure (NYCOMPS), New York Structural Biology Center (NYSBC), New York
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 29.6
DOI:  10.1002/0471140864.ps2906s74
Online Posting Date:  November, 2013
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Abstract

Recently, several structural genomics centers have been established and a remarkable number of three‐dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over‐expression and purification of membrane proteins is a much more arduous process. By using high‐throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression, and screening of membrane proteins using high‐throughput methodologies developed in the laboratory. Basic Protocol 1 describes cloning of inserts into expression vectors by ligation‐independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that do express on the miniscale, Basic Protocols 3 and 4 outline the methods employed for the expression and purification of targets on a midi‐scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. Curr. Protoc. Protein Sci. 74:29.6.1‐29.6.34. © 2013 by John Wiley & Sons, Inc.

Keywords: Escherichia coli; high‐throughput; cloning/expression; recombinant protein expression; membrane protein; ligation‐independent cloning (LIC); membrane protein purification

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: High‐Throughput Cloning of Open Reading Frames Encoding Integral Membrane Proteins into E. coli Expression Vectors
  • Basic Protocol 2: Small‐Scale Expression and Purification
  • Basic Protocol 3: Mid‐Scale Expression of 96 Proteins in the GNF Fermenter
  • Alternate Protocol 1: Mid‐Scale Expression Using Erlenmeyer Flasks
  • Basic Protocol 4: Mid‐Scale Purification and Size‐Exclusion Chromatography
  • Alternate Protocol 2: Detergent Stability Assay
  • Support Protocol 1: Digestion and Gel Purification of Vector for LIC Cloning
  • Support Protocol 2: PCR Amplification and Purification of PCR Products
  • Support Protocol 3: Robotic Isolation of Plasmid DNA
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: High‐Throughput Cloning of Open Reading Frames Encoding Integral Membrane Proteins into E. coli Expression Vectors

  Materials
  • Expression vector
  • Restriction enzymes
  • 10× buffer 2 (New England Biolabs):
    • 10 mM Tris·Cl, pH 7.9
    • 50 mM NaCl
    • 10 mM MgCl 2
    • 1 mM DTT
  • 100× bovine serum albumin (BSA) (10 mg/ml; New England Biolabs)
  • Deoxynucleotides (100 mM; Invitrogen)
  • T4 DNA polymerase (New England Biolabs, cat. no. M0203)
  • 25 mM EDTA
  • E. coli DH10B‐T1R competent cells
  • SOC medium (see recipe), prewarmed
  • LB‐agar (see recipe)
  • Antibiotics
  • 96‐well PCR plates (VWR, cat. no. 82006‐704 or Eppendorf, cat. no. 951020401)
  • Thermal cycler
  • 384‐well PCR plates (Eppendorf, cat. no. 951020702)
  • Multichannel pipettor
  • Adhesive‐backed porous seals (VWR, cat. no. 60941‐086)
  • 37°C shaking incubator (Shel Lab, cat. no. SI6R‐HS or New Brunswick Innova 44)
  • 24‐well blocks (Qiagen, cat. no. 19583)
  • Glass beads (Sigma, cat. no. Z273627)

Basic Protocol 2: Small‐Scale Expression and Purification

  Materials
  • Miniprep plasmid DNA
  • E. coli BL21(DE3)‐pLysS‐T1R competent cells
  • 2× TY medium (see recipe)
  • Antibiotics
  • 2× glycerol storage solution (see recipe)
  • 100 mM isopropyl‐β‐D‐1‐thiogalactopyranoside (IPTG)
  • Resuspension buffer (see recipe)
  • Benzonase nuclease (EMD Millipore, cat. no. 70664‐250KUN)
  • 4‐(2‐Aminoethyl)‐benzenesulfonyl fluoride HCl (AEBSF, Bio‐Research Products, cat. no. 401)
  • Resuspension buffer containing 12% (w/v) N‐dodecyl‐β‐D‐maltopyranoside (DDM, Affymetrix, cat. no. D310)
  • Nickel‐chelating resin (GE Healthcare, Qiagen, G Biosciences, and others)
  • Wash buffer B (see recipe)
  • Elution buffer (see recipe)
  • 5× SDS‐PAGE loading buffer (see recipe)
  • Criterion Precast 4% to 20% (w/v) acrylamide‐Tris·Cl gels, 26 wells (Bio‐Rad, cat. no. 345‐0034)
  • Coomassie blue
  • 96‐well PCR plates
  • Thermal cycler
  • 96‐well deep‐well blocks (Costar, cat. no. 3960)
  • 37°C shaking incubator (Shel Lab, cat. no. SI6R‐HS)
  • Adhesive‐backed porous (VWR, cat. no. 60941‐086) and foil (VWR, cat. no. 60941‐076) seals
  • Spectrophotometer
  • Centrifuge with plate rotor
  • Sonicator robot‐small probe (ST Robotics), 30% maximum amplitude (or hand‐held sonicator)
  • Titramax 1000 shaking platform (Heidolph Instruments), 4°C
  • 50‐ml centrifuge tubes
  • 96‐well, 2‐ml filter plates (Thompson Instrument Company, cat. no. 931919)
  • 96‐well bottom plate seal (Thompson Instrument Company, cat. no. 982005)
  • Large orifice pipet tips
  • Vacuum manifold
  • 96‐well U‐bottomed plates (Greiner, cat. no. 650101)

Basic Protocol 3: Mid‐Scale Expression of 96 Proteins in the GNF Fermenter

  Materials
  • 2× TY medium (see recipe)
  • Antibiotics
  • Frozen glycerol stocks (see protocol 1)
  • Terrific broth (TB, see recipe)
  • 1 M 4‐morpholinopropanesulfonic acid (MOPS), pH 7.6 (see recipe)
  • 1 M MgSO 4
  • Compressed air and bottled O 2
  • 1 M isopropyl‐β‐D‐1‐thiogalactopyranoside (IPTG)
  • 96‐well deep‐well blocks
  • Hot plate
  • Cryo‐replicator (EnzyScreen, cat. no. CR1000)
  • 37°C shaker incubator (Shel Lab)
  • GNF Fermenter (Genomics Institute of the Novartis Research Foundation)
  • Liquid handling robot (Beckman Coulter Biomek FX)
  • Innova shaker
  • Peristaltic pump
  • Gas manifold
  • 50‐ml conical tubes
  • Centrifuge

Alternate Protocol 1: Mid‐Scale Expression Using Erlenmeyer Flasks

  Materials
  • Glycerol stocks (see protocol 1)
  • 2× TY medium (see recipe)
  • Antibiotics
  • 1 M isopropyl‐β‐D‐1‐thiogalactopyranoside (IPTG)
  • Antifoam 204 (Sigma A6426)
  • 50‐ml conical centrifuge tubes
  • 37°C shaking incubator containing 50‐ml tube rack
  • 2‐liter baffled Erlenmeyer flasks
  • Innova 44 incubator (Eppendorf)
  • Centrifuge

Basic Protocol 4: Mid‐Scale Purification and Size‐Exclusion Chromatography

  Materials
  • Frozen cell pellets
  • Resuspension buffer (see recipe)
  • Benzonase nuclease (EMD Millipore, cat. no. 70664‐250KUN)
  • 4‐(2‐Aminoethyl)‐benzenesulfonyl fluoride HCl (AEBSF, Bio‐Research Products, cat. no. 401)
  • Resuspension buffer containing 12% (w/v) DDM (see recipe)
  • Nickel‐chelating resin (Ni2+, GE Healthcare, Qiagen, G Biosciences, and others)
  • ATP wash buffer (see recipe)
  • Wash buffer B (see recipe)
  • Elution buffer (see recipe)
  • 2× SDS‐PAGE loading buffer (see recipe)
  • Criterion precast 4% to 20% (w/v) acrylamide/Tris·Cl gels, 26 wells (BioRad, cat. no. 345‐0034)
  • Coomassie Blue
  • 50‐ml conical centrifuge tubes
  • Sonicator robot‐large probe (ST Robotics), 60% maximum amplitude (or a hand‐held sonicator)
  • Roto‐Shake Genie (VWR, cat. no. 58815‐176; or equivalent rotating platform)
  • Refrigerated centrifuge (Beckman Coulter Avanti J26‐XP or equivalent)
  • 96‐well, 2‐ml filter plates (Thompson Instrument Company, cat. no. 931919)
  • 96‐well bottom plate seal (Thompson Instrument Company, cat. no. 982005)
  • Titramax 1000 shaking platform (Heidolph Instruments)
  • Vacuum manifold
  • 96‐well U‐bottomed plates (Greiner, cat. no. 650101)
  • HPLC system (Agilent Technologies 1200 Series)
  • TSK‐gel Super SW3000 size‐exclusion column, 4.6 × 300–mm (Tosoh, cat. no. 18675) or 5/150 Superdex S200 column (GE Healthcare, cat. no. 28‐9065‐61)
  • Spectrophotometer

Alternate Protocol 2: Detergent Stability Assay

  Materials
  • Eluate (sample from protocol 5, step 18, in 200 µl elution buffer)
  • N‐dodecyl‐β‐D‐maltopyranoside (DDM, Affymetrix)
  • n‐Dodecyl‐N,N‐dimethylamine‐N‐oxide (LDAO, Affymetrix)
  • 2‐[2‐[2‐(2‐octoxyethoxy)ethoxy]ethoxy]ethanol (C 8E 4, Affymetrix)
  • n‐Octyl‐β‐D‐glucopyranoside (βOG, Affymetrix)
  • Gel filtration buffer (see recipe)
  • 96‐well U‐bottom plates (Greiner, cat. no. 650101)
  • Adhesive‐backed foil (VWR, cat. no. 60941‐076)
  • 384‐well filter plates (Pall, cat. no. 5070)
  • 384‐well receiver plates (Nunc, cat. no. 264573)
  • Centrifuge with plate rotor
  • 96‐well plates for HPLC (Agilent, cat. no. 5042‐8502 or Eppendorf, cat. no. 951020401)
  • Pierceable silicone sealing mats (Agilent, cat. no. 521‐01‐151)
  • HPLC system (Agilent Technologies 1200 Series)
  • TSK‐Gel Super SW3000 size exclusion column, 4.6 × 300–mm (Tosoh, cat. no. 18675) or 5/150 Superdex S200 column (GE Healthcare, cat. no. 28‐9065‐61)
  • Spectrophotometer

Support Protocol 1: Digestion and Gel Purification of Vector for LIC Cloning

  Materials
  • Plasmid DNA sample
  • 100× BSA (10 mg/ml)
  • Restriction enzymes SnaBI, BfuAI, SspI, and SmaI and 10× buffer (New England Biolabs)
  • 0.8% to 1.0% agarose gel
  • Gel extraction kit (Qiagen, cat. no. 28706) or equivalent
  • 25°C, 37°C, 50°C, and 65°C incubators
  • Agarose gel electrophoresis system

Support Protocol 2: PCR Amplification and Purification of PCR Products

  Materials
  • KOD Hot‐Start DNA polymerase kit (Novagen, cat. no. 71086) containing:
    • KOD Hot‐Start DNA polymerase
    • 10× PCR buffer
    • 25 mM MgSO 4
    • dNTP mix (2 mM each)
  • Template DNA (genomic DNA at 10 ng/µl, plasmid DNA at 0.5 ng/µl, cDNA library at 2 ng/µl, etc.)
  • Sense and anti‐sense primer (5 µM each) assembled in 384‐ or 96‐well plates
  • Dimethyl sulfoxide (DMSO), optional
  • DpnI restriction enzyme (New England Biolabs), optional
  • 10× buffer 4 (New England Biolabs):
    • 20 mM Tris‐acetate, pH 7.9
    • 50 mM potassium acetate
    • 10 mM magnesium acetate
    • 1 mM DTT
  • AMPure XP PCR purification kit (Beckman Coulter, cat. no. A63880)
  • 96‐well E‐gel 2% (w/v) agarose (Invitrogen, cat. no. G7008‐02)
  • Liquid handling robot
  • 96‐well PCR plates (Eppendorf, cat. no. 951020401) or 384‐well PCR plates (Eppendorf, cat. no. 951020702)
  • Adhesive foil for microplates (VWR, cat. no. 60941‐076)
  • Thermal cycler (Eppendorf Mastercycler, Eppendorf Mastercycler ep384, or similar)
  • 96‐well silicone sealing mat (Axygen, cat. no. CM‐96‐RD)
  • Mother E‐Base (Invitrogen, cat. no. EB‐M03)

Support Protocol 3: Robotic Isolation of Plasmid DNA

  Materials
  • Colonies grown in 24‐well blocks (see protocol 1, step 11)
  • 2× TY medium (see recipe)
  • Antibiotics
  • CosMCPrep plasmid DNA isolation kit (Beckman Coulter, cat. no. A29174)
  • Sterile toothpicks
  • 96‐well deep‐well blocks (Costar, cat. no. 3960)
  • 37°C shaking incubator (Shel Lab)
  • Centrifuge with plate rotor
  • Liquid handling robot
  • 96‐well round‐bottom plates (Greiner, cat. no. 650101)
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Figures

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Key References
  Graslund et al., 2008. See above.
  These two reviews describe the different cloning, expression, and purification methods employed for high‐throughput protein production. The former also summarizes the different approaches used by structural genomics centers.
  Stevens, 2000. See above.
  Reviews recent progress of IMP structure determination by structural genomics (SG) initiatives and discusses the progress of SG centers in covering important protein families.
  Kloppmann et al., 2012. See above.
  Describes target selection of bacterial αIMPs for the NYCOMPS high‐throughput expression pipeline and discusses the potential impact of structure determination of these targets on a larger uncharacterized pool of proteins.
  Punta et al., 2009. See above.
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