Obtaining Soluble Folded Proteins from Inclusion Bodies Using Sarkosyl, Triton X‐100, and CHAPS: Application to LB and M9 Minimal Media

Michael A. Massiah1, Katharine M. Wright1, Haijuan Du1

1 Department of Chemistry, George Washington University, Washington, D.C
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 6.13
DOI:  10.1002/0471140864.ps0613s84
Online Posting Date:  April, 2016
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Abstract

This unit describes a straightforward and efficient method of using sarkosyl to solubilize and recover difficult recombinant proteins, such as GST‐ and His6‐tagged fusion proteins, that are overexpressed in E. coli. This protocol is especially useful for rescuing recombinant proteins overexpressed in M9 minimal medium. Sarkosyl added to lysis buffers helps with both protein solubility and cell lysis. Higher percentage sarkosyl (up to 10%) can extract >95% of soluble protein from inclusion bodies. In the case of sarkosyl‐solubilized GST‐fusion proteins, batch‐mode affinity purification requires addition of a specific ratio of Triton X‐100 and CHAPS, while sarkosyl‐solubilized His6‐tagged fusion proteins can be directly purified on Ni2+ resin columns. Proteins purified by this method could be widely used in biological assays, structure analysis and mass spectrum assay. © 2016 by John Wiley & Sons, Inc.

Keywords: sarkosyl; Inclusion bodies; protein expression and purification; Triton X‐100; CHAPS; M9 minimal medium; Glutathione S‐transferase (GST); His6‐tagged

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Expression and Purification of Large‐Scale Glutathione‐S‐Transferase Fusion Protein with Sarkosyl
  • Basic Protocol 2: Expression and Purification of His6‐Tagged E. coli Fusion Protein with Sarkosyl
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Expression and Purification of Large‐Scale Glutathione‐S‐Transferase Fusion Protein with Sarkosyl

  Materials
  • Sterile LB Agar plates (see recipe)
  • 100 mg/ml stock ampicillin (see recipe)
  • Glycerol stock of transformed E.coli cells expressing a GST‐fusion protein in pGEX‐4 T‐2 vector (see Strategic Planning)
  • Sterile LB medium, pH 7.2 (see recipe)
  • 440 mM sorbitol
  • 2.5 mM betaine
  • Isopropyl‐1‐thio‐β‐D‐galactopyranoside (IPTG), 1 M stock (see recipe)
  • Sterile M9 minimal medium, pH 7.4 (see recipe)
  • Ice
  • Lysis buffer (see recipe), ice cold
  • 100 mg/ml lysozyme (see recipe)
  • 10 mg/ml Deoxyribonuclease I (DNase I; see recipe)
  • N‐laurylsarcosine Sarkosyl, 30% (w/v) stock (see recipe)
  • Glutathione‐Sepharose 4B resin (GE Healthcare, cat. no. 17‐5732‐01)
  • Q‐water
  • Triton X‐100, 30% (v/v) stock (see recipe)
  • CHAPS, 0.4 M stock (see recipe)
  • Binding buffer (see recipe)
  • Imidazole, 2.5 M stock
  • Glutathione buffer (see recipe)
  • Wash buffer (see recipe), ice cold
  • Bradford assay dye reagent concentrate (BioRad, cat. no. 500‐0006)
  • Inoculating loop
  • Mini incubator, 37°C
  • Sterilized toothpicks, optional
  • 50‐ml centrifuge tubes
  • Refrigerated environmental shaker (e.g., New Brunswick Scientific Excella E25)
  • High‐ and low‐speed refrigerated centrifuge (e.g., Thermo Scientific Sorvall 6000), 4°C
  • Vortex mixer
  • 2‐liter Erlenmeyer baffled cell culture flasks
  • UV/Visible light spectrophotometer with cuvettes
  • Large 1‐liter centrifuge bottles
  • Serological pipets
  • Balance
  • Benchtop gyroscopic mixer/rocker
  • Probe‐tip Sonicator (e.g., Fischer Sonic Dismembrator 550)
  • 30‐ml centrifuge bottles (capable of handling g‐force of 48,000 × g)
  • High‐speed SS‐34 rotor
  • Transfer pipets
  • 15‐ml conical tubes
  • 0.7‐ and 1.7‐ml microcentrifuge tubes
  • NOTE: Ampicillin should be used as the selection antibiotic with the pGEX‐4T2 plasmid.
  • Additional reagents and equipment for SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE)

Basic Protocol 2: Expression and Purification of His6‐Tagged E. coli Fusion Protein with Sarkosyl

  Materials
  • Sterile LB agar plates (see recipe)
  • 30 mg/ml stock Kanamycin (see recipe)
  • Glycerol stock of transformed E.coli cells expressing a His 6‐tagged protein in T7 vector (see Strategic Planning)
  • Sterile LB medium, pH 7.2 (see recipe)
  • Isopropyl‐1‐thio‐β‐D‐galactopyranoside (IPTG), 1 M stock (see recipe)
  • Sterile M9 minimal medium, pH 7.4 (see recipe)
  • Ice
  • Lysis buffer/Binding buffer/Wash buffer (see recipe)
  • Sarkosyl, 30% (w/v) stock (see recipe)
  • Ni2+‐NTA Agarose Resin (G‐Biosciences, cat. no. 786‐407)
  • Q‐water (18 mΩ resistance)
  • Imidazole, 2.5 M stock, pH 7.7
  • Bradford assay dye reagent concentrate (BioRad, cat. no. 500‐0006)
  • Triton X‐100, 30% (v/v) stock (see recipe)
  • CHAPS, 0.4 M stock (see recipe)
  • Sterilized toothpick or inoculating loop
  • 50‐ml high‐speed‐compatible centrifuge tubes
  • Refrigerated environmental shaker
  • 2‐liter Erlenmeyer baffled cell culture flasks
  • UV/Visible light spectrophotometer with cuvettes
  • Large 1‐liter centrifuge bottles
  • Refrigerated centrifuge
  • Serological pipets
  • Mini incubator, 37°C
  • Vortex mixer
  • Transfer pipets
  • Benchtop Orbital Shaker
  • Probe‐tip Sonicator (e.g., Fischer Sonic Dismembrator 550)
  • Plastic columns (30 ml volume)
  • 0.7‐ and 1.7‐ml microcentrifuge tubes
NOTE: Kanamycin antibiotic should be used with the pETite plasmid.
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Figures

Videos

Literature Cited

Literature Cited
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  Tao, H., Liu, W., Simmons, B., Harris, H., Cox, T., and Massiah, M. 2010. Purifying natively folded proteins from inclusion bodies using sarkosyl, Triton X‐100, and CHAPS. Biotechniques 48:61‐64. doi: 10.2144/000113304.
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Key References
  Burgess, 2009. See above.
  Tao et al., 2010. See above.
  Waugh, 2005. See above.
Internet Resources
  www.lucigen.com
  For pETite His6‐tag: (Manuals: Expresso T7 Cloning and Expression System).
  www.gelifesciences.com
  For pGEX‐4T2 GST‐fusion: (Handbooks: GST Gene Fusion System).
  www.bioinformatics.org
  For primer design: Sequence manipulation Suite: PCR Primer Stats.
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