Folding and Purification of Insoluble (Inclusion Body) Proteins from Escherichia coli

Paul T. Wingfield1, Ira Palmer1, Shu‐Mei Liang2

1 Protein Expression Laboratory, NIAMD/NIH, Bethesda, Maryland, 2 Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 6.5
DOI:  10.1002/0471140864.ps0605s78
Online Posting Date:  November, 2014
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Abstract

Heterologous expression of recombinant proteins in E. coli often results in the formation of insoluble and inactive protein aggregates, commonly referred to as inclusion bodies. To obtain the native (i.e., correctly folded) and hence active form of the protein from such aggregates, four steps are usually followed: (1) the cells are lysed, (2) the cell wall and outer membrane components are removed, (3) the aggregates are solubilized (or extracted) with strong protein denaturants, and (4) the solubilized, denatured proteins are folded with concomitant oxidation of reduced cysteine residues into the correct disulfide bonds to obtain the native protein. This unit features three different approaches to the final step of protein folding and purification. In the first, guanidine·HCl is used as the denaturant, after which the solubilized protein is folded (before purification) in an “oxido‐shuffling” buffer system to increase the rate of protein oxidation. In the second, acetic acid is used to solubilize the protein, which is then partially purified by gel filtration before folding; the protein is then folded and oxidized by simple dialysis against water. Thirdly, folding and purification of a fusion protein using metal‐chelate affinity chromatography are described. © 2014 by John Wiley & Sons, Inc.

Keywords: protein folding; protein purification; recombinant protein; Escherichia coli; inclusion body

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Folding and Purification of Bovine Growth Hormone
  • Support Protocol 1: Direct Determination of the Molecular Weight of BGH Using Sedimentation Equilibrium
  • Basic Protocol 2: Folding and Purification of Human Interleukin 2
  • Support Protocol 2: Resolution of Native and Misfolded Forms of hIL‐2 by RP‐HPLC
  • Basic Protocol 3: Folding and Purification of a Histidine‐Tagged Protein: HIV‐1 Integrase
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Folding and Purification of Bovine Growth Hormone

  Materials
  • E. coli cells expressing BGH: ∼50 g wet weight from a 1.5‐liter fermentation (unit 5.3), and stored as a flattened paste in a sealed polyethylene bag at −80°C
  • BGH break buffer A (see recipe), 4°C
  • BGH break buffer B (see recipe), 4°C
  • BGH wash buffer A (see recipe), 4°C
  • BGH wash buffer B (see recipe), 4°C
  • BGH extraction buffer (see recipe), 4°C
  • BGH folding buffer A (see recipe), 4°C
  • BGH folding buffer B (see recipe), 4°C
  • 2 M HCl
  • BGH column buffer A (see recipe), 4°C
  • DEAE Sepharose CL‐4B ion‐exchange resin (GE Healthcare)
  • Sephadex G‐100 gel filtration resin (GE Healthcare)
  • BGH column buffer B (see recipe), 4°C
  • Blender (e.g., Waring; ≥1 liter capacity)
  • Tissue homogenizer (e.g., Polytron: Brinkmann or equivalent)
  • Beckman J2‐21M centrifuge and JA‐20 rotor (or equivalent centrifuge: Beckman Coulter)
  • Sonicator, ≥400 W, with sound enclosure (Branson or equivalent)
  • 0.7‐μm glass‐microfiber filters, 4.7‐cm diameter (GE Healthcare—Whatman GF/F) with vacuum filtration apparatus (optional)
  • Spectra/Por 1 dialysis tubing, 40‐mm diameter (MWCO 6000 to 8000; Spectrum Labs)
  • 5 × 50–cm and 5 × 100–cm glass chromatography columns with adjustable flow adaptors (GE Healthcare)
  • Stirred cell with Diaflo PM 10 ultrafiltration membrane (EMD Millipore)
  • Millex‐GV 0.22‐μm filter units (EMD Millipore)
  • Additional reagents and equipment for cell breakage using a French press (unit 6.2), dialysis ( appendix 3B), ion‐exchange chromatography (unit 8.2), gel‐filtration chromatography (unit 8.3), and SDS‐PAGE (unit 10.1)

Support Protocol 1: Direct Determination of the Molecular Weight of BGH Using Sedimentation Equilibrium

  Additional Materials ( )
  • BGH from peak fractions in column buffer B ( protocol 1, step 13)
  • Beckman Optima XL‐I analytical ultracentrifuge
  • ProteomeLab XL‐A/XL‐I (previously known as Optima XL‐A/XL‐1) (Beckman Coulter)
  • Analytical Ti Rotor An‐60 4‐place (8‐place also available) (Beckman Coulter)
  • Analytical cell assembly with quartz windows (Beckman Coulter)
  • Standard double‐sector cells (cells and other supplies compatible with the XL‐A/I can be obtained from Spin Analytical, http://www.spinanalytical.com/)
  • Beckman XL‐A/ XL‐I data analysis software V6.04 (runs using Origin V6.0)

Basic Protocol 2: Folding and Purification of Human Interleukin 2

  Materials
  • hIL‐2 break buffer (see recipe), 4°C
  • E. coli cells expressing hIL‐2: ∼20 g wet weight from a 3‐liter fermentation (unit 5.3), and stored as a flattened paste in a sealed polyethylene bag at –80°C
  • Sucrose
  • Lysozyme (Worthington)
  • hIL‐2 wash buffer: 0.75 M guanidine·HCl/1% (w/v) Tween 20 (prepare immediately before use), 4°C
  • PBS ( appendix 2E), 4°C
  • 10% and 20% (v/v) acetic acid, 4°C (prepare fresh from glacial acetic acid)
  • Sephadex G‐100 gel‐filtration resin (GE Healthcare)
  • Acetonitrile (HPLC grade)
  • Trifluoroacetic acid (TFA; HPLC grade)
  • 7 × 250–mm 300‐Å octyl Aquapore RP‐300 semiprep column (Perkin Elmer)
  • RP‐HPLC solvent A (see recipe), room temperature
  • RP‐HPLC solvent B (see recipe), room temperature
  • 25 mM acetic acid, 4°C
  • Tissue homogenizer (e.g., Polytron, Brinkmann)
  • 30°C water bath
  • Sorvall RC‐5C centrifuge with SS‐34 rotor (or equivalent from Beckman Coulter)
  • 2.6 × 100–cm glass chromatography column (see GE Healthcare for column selection)
  • Spectra/Por 3 dialysis tubing, 11.5‐ and 45‐mm diameters (MWCO 3500; Spectrum Labs)
  • Sterivex‐GS 0.22‐μm filter units (EMD Millipore)
  • HPLC system with pumps, UV detector, and fraction collector (Waters or equivalent manufacturer)
  • Additional reagents and equipment for cell breakage using a French press (unit 6.2), gel‐filtration chromatography (unit 8.3), and dialysis ( appendix 3B)

Support Protocol 2: Resolution of Native and Misfolded Forms of hIL‐2 by RP‐HPLC

  Additional Materials (also see protocol 3)
  • 0.46 × 10–cm SynChropak RP‐P C18 column (or equivalent RP C18 column from Agilent Technologies)
  • RP‐HPLC solvent C (see recipe)
  • 25 mM acetic acid
  • Purified folded hIL‐2 solution (see protocol 3)

Basic Protocol 3: Folding and Purification of a Histidine‐Tagged Protein: HIV‐1 Integrase

  Materials
  • E. coli cells expressing HIV‐1 integrase: 100 g wet weight from a 3.5‐liter fermentation (unit 5.3), stored as a flattened paste in a sealed polyethylene bag at −80°C
  • IN break buffer (see recipe), 4°C
  • Suspension buffer: IN break buffer (see recipe) prepared without lysozyme, 4°C
  • IN extraction buffer (see recipe), 4°C
  • 6 × 60–cm Superdex 200 prep grade prepacked gel‐filtration column (Pharmacia Biotech)
  • IN column buffer A (see recipe), 4°C
  • Ni‐NTA‐Sepharose CL‐6B MCAC resin (Qiagen; GE Healthcare and other suppliers)
  • IN column buffer B (see recipe), 4°C
  • IN column buffer C (see recipe), 4°C
  • IN column buffer D (see recipe), 4°C6 × 60–cm Superdex 75 prep grade prepacked gel‐filtration column (GE Healthcare)
  • IN column buffer E (see recipe), 4°C
  • 4 M guanidine·HCl/5 mM DTT (4°C)
  • IN folding buffer (see recipe), 4°C
  • 50 mM Tris·Cl (pH 7.5 at 4°C)/10 mM CHAPS, 4°C
  • 2000 NIH unit/ml thrombin (see recipe)
  • IN column buffer F (see recipe), 4°C
  • 9 M urea, 4°C
  • IN column buffer G (see recipe), 4°C
  • p‐aminobenzamidine immobilized on Sepharose 6B (GE Healthcare; Sigma Aldrich)
  • 0.1 mM 4‐(2‐aminoethyl)benzenesulfonyl fluoride (AEBSF: Sigma Aldrich, Thermo Scientific, and others)
  • Blender (e.g., Waring; ≥1 liter capacity)
  • 1‐liter steel beaker
  • Sonicator, ≥400 W, with sound enclosure (Branson or equivalent)
  • Centrifuge with Beckman J‐14 rotor (or equivalent)
  • Tissue homogenizer (e.g., Polytron, Brinkmann)
  • Ultracentrifuge with Beckman 45Ti rotor (or equivalent)
  • 5 × 50–cm chromatography column with adjustable flow adaptors
  • Stirred cells (400‐ml and 2‐liter capacities) with Diaflo PM 10 ultrafiltration membranes (EMD Millipore)
  • Peristaltic pump
  • Millex‐GV 0.22‐μm pore size filter units (EMD Millipore)
  • 28°C water bath
  • 1 × 10– to 1 × 20–cm chromatography column
  • Additional reagents and equipment for gel‐filtration chromatography (unit 8.3) and SDS‐PAGE (unit 10.1)
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Figures

Videos

Literature Cited

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