Production of Disulfide‐Bonded Proteins in Escherichia coli

Na Ke1, Mehmet Berkmen1

1 New England Biolabs, Ipswich, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.1B
DOI:  10.1002/0471142727.mb1601bs108
Online Posting Date:  October, 2014
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Abstract

Production of recombinant proteins at high yields in Escherichia coli requires extensive optimization of expression conditions. Production is further complicated for proteins that require specific post‐translational modifications for their eventual folding. One common and particularly important post‐translational modification is oxidation of the correct pair of cysteines to form a disulfide bond. This unit describes methods to produce disulfide‐bonded proteins in E. coli in either the naturally oxidizing periplasm or the cytoplasm of appropriately engineered cells. The focus is on variables key to improving the oxidative folding of disulfide‐bonded proteins, with the aim of helping the researcher optimize expression conditions for a protein of interest. Curr. Protoc. Mol. Biol. 108:16.1B.1‐16.1B.21. © 2014 by John Wiley & Sons, Inc.

Keywords: disulfide‐bonded protein production; E. coli ; SHuffle

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Using Alkylation to Determine the Oxidation State of Cysteines
  • Basic Protocol 2: Expression and Extraction of Disulfide‐Bonded Proteins in E. coli Cytoplasm
  • Basic Protocol 3: Extraction of Disulfide‐Bonded Proteins from E. coli Periplasm
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Using Alkylation to Determine the Oxidation State of Cysteines

  Materials
  • E. coli strains (e.g., DHB4 [wt E. coli K12], MB68 [DHB4 ΔdsbA], MB69 [DHB4 ΔdsbC]) expressing pAppA (pBAD18‐pAppA; Berkmen et al., )
  • NZ medium (see recipe) containing antibiotics (e.g., 10 μg/ml chloramphenicol)
  • 20% (w/v) arabinose
  • 100% (w/v) TCA (see recipe)
  • Acetone (Sigma‐Aldrich, cat. no. 650501), ice cold
  • 100 mM Tris‐Cl, pH 8.0 ( )
  • Sodium dodecyl sulfate (SDS)
  • Dithiothreitol (DTT)
  • AMS alkylation buffer (see recipe)
  • 100 mM Tris·Cl, pH 6.8 ( ) containing 1% SDS
  • 3× SDS Blue Loading Buffer (New England Biolabs, cat. no. B7703S)
  • Anti‐AppA serum (Berkmen et al., )
  • 1.5‐ml microcentrifuge tubes
  • Analog vortex mixer with microtube adapter for single tube holder and flat foam insert (VWR, cat. nos. 58816‐121, 12620‐894, and 12620‐884)
  • Additional reagents and equipment for SDS‐PAGE and immunoblotting (units & )

Basic Protocol 2: Expression and Extraction of Disulfide‐Bonded Proteins in E. coli Cytoplasm

  Materials
  • Anti‐MBP IgG expression plasmid (pET21b‐cIgG, Novagen)
  • E. coli strains (New England Biolabs): SHuffle T7 Express (E. coli B, cat. no. C3029) and SHuffle T7 (E. coli K12, cat. no. C3026)
  • NZ medium (see recipe) containing 10 μg/ml ampicillin
  • 1 M IPTG (see recipe)
  • Lysis buffer (see recipe)
  • 10 μg/ml maltose‐binding protein (MBP) in PBS (see recipe for PBS)
  • PBST: PBS (see recipe) with 0.1% Tween 20
  • Blocking buffer: PBS (see recipe) with 1% BSA
  • Anti‐mouse IgG (Cell Signaling Technology, cat. no. 7076)
  • 1‐Step Ultra TMB ELISA substrate solution (Thermo Scientific, cat. no. 34028)
  • 2 M H 2SO 4
  • 250‐ml flask
  • Low‐temperature incubator (30° and 16°C)
  • 30‐ml centrifuge tubes
  • 1.5‐ml microcentrifuge tubes
  • Ultrasonic liquid processor (Qsonica, XL‐2000 Microson)
  • ELISA plates (ImmunoChemistry Technologies, cat. no. 227)
  • Flatbed platform shaker
  • Microplate reader (SpectraMax M5, Molecular Devices)

Basic Protocol 3: Extraction of Disulfide‐Bonded Proteins from E. coli Periplasm

  Additional Materials protocol 2)
  • Cell pellet containing periplasmic expression of a protein of interest (see protocol 2, steps 1‐5)
  • 30 mM Tris·Cl, pH 8.0, with 20% sucrose
  • 0.5 M EDTA pH 8.0
  • 5 mM MgSO 4, ice cold
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Figures

Videos

Literature Cited

Literature Cited
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