Preparation and Extraction of Insoluble (Inclusion‐Body) Proteins from Escherichia coli

Ira Palmer1, Paul T. Wingfield1

1 National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 6.3
DOI:  10.1002/0471140864.ps0603s70
Online Posting Date:  November, 2012
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High‐level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies. Inclusion bodies are normally formed in the cytoplasm; however, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies can be recovered from cell lysates by low‐speed centrifugation. Following pre‐extaction (or washing), protein is extracted from washed pellets using guanidine⋅HCl. The solubilized and unfolded protein is either directly folded or further purified by gel filtration in the presence of guanidine⋅HCl as described in this unit. A support protocol describes the removal of guanidine⋅HCl from column fractions so they can be monitored by SDS‐PAGE. Curr. Protoc. Protein Sci. 70:6.3.1‐6.3.20. © 2012 by John Wiley & Sons, Inc.

Keywords: recombinant protein; Escherichia coli; inclusion bodies; protein extraction; protein solubilization; guanidine⋅HCl; protein purification; cell lysis

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation and Extraction of Insoluble (Inclusion‐Body) Proteins from Escherichia coli
  • Basic Protocol 2: Medium‐Pressure Gel‐Filtration Chromatography in the Presence of Guanidine Hydrochloride
  • Support Protocol 1: Preparation of Samples Containing Guanidine Hydrochloride for SDS‐PAGE
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Preparation and Extraction of Insoluble (Inclusion‐Body) Proteins from Escherichia coli

  • E. coli cells from fermentation (unit 5.3) containing the protein of interest
  • Lysis buffer (see recipe)
  • Ice
  • Wash buffer (see recipe), with and without urea and Triton X‐100
  • Extraction buffer (see recipe)
  • 250‐ and 500‐ml stainless steel beakers
  • Ice bucket
  • Waring blender
  • Polytron tissue‐grinder homogenizer (Brinkmann; http://www.kinematica‐
  • French pressure cell (e.g., Thermo Electron Corp;
  • Probe sonicator
  • Beckman J2‐21M centrifuge with JA‐14 rotor (or equivalent)
  • Beckman Optima XL‐90 ultracentrifuge with 45 Ti rotor (or equivalent)
  • 0.22‐µm syringe filters (e.g., Millex from Millipore)
  • 20‐ml disposable syringe
  • Additional equipment for breaking cells, homogenizing cells and pellets and centrifuging at low and high speeds (unit 6.2)

Basic Protocol 2: Medium‐Pressure Gel‐Filtration Chromatography in the Presence of Guanidine Hydrochloride

  • Gel‐filtration medium: Superdex 200 PG (preparative grade; GE Healthcare Life Sciences)
  • 5% (v/v) ethanol
  • Gel‐filtration buffer (see recipe)
  • Guanidine⋅HCl extract of E. coli cells containing the protein of interest (see protocol 1)
  • 4‐ to 6‐liter plastic beaker
  • Chromatography column: GE Healthcare Life Sciences XK 16/100, 26/100, or 50/100
  • Glass rod or plastic paddle
  • Vacuum flask and laboratory vacuum
  • Packing reservoir: GE Healthcare Life Sciences RK 16/26 (for 16‐ and 26‐mm‐i.d. columns) and RK 50 (for 50‐mm‐i.d. column)
  • Chromatography pump: GE Healthcare Life Sciences P‐50 or P‐900
  • Injection valve (to select between sample loop and pump)
  • UV monitor and fraction collector
  • Sample loop (volume determined by size of column; also see annotation to step 15)
NOTE: The various components of the chromatography system (pumps, valves, monitors, and sample loops) listed separately above are supplied as components of the ÄKTAexplorer chromatography system (GE Healthcare Life Sciences), which is used to run the XK 50/100 column. The smaller XK columns (2.6 and 2.5 cm i.d.) are run using the ÄKTA‐FPLC chromatography system (also from GE Healthcare Life Sciences), which is designed for small‐ to medium‐scale work. For further details on this equipment, see the manufacturer's literature (e.g., Process Products, GE Healthcare Life Sciences).NOTE: Perform steps 1 to 11 at room temperature. After the column is packed, equilibrate and elute at 4°C.

Support Protocol 1: Preparation of Samples Containing Guanidine Hydrochloride for SDS‐PAGE

  • Sample containing the protein of interest
  • 100% and 90% ethanol, 0° to 4°C
  • 1× SDS sample buffer (unit 10.1)
  • Gilson Pipetman (
  • 1.5‐ml microcentrifuge tubes
  • Vortex mixer
  • Microcentrifuge
  • Additional reagents and equipment for gel electrophoresis (unit 10.1)
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  •   FigureFigure 6.3.1 Analysis by SDS‐PAGE of fractions from low‐speed centrifugation of E. coli cell lysates containing aggregated bovine growth hormone. A 12.5% acrylamide gel of dimensions 12 cm × 16 cm × 1.5 mm was used with the Laemmli buffer system ( UNIT). Lanes a and g contain molecular weight standards (low‐range standards, Bio‐Rad) in order of increasing migration distance: phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), hen egg white ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and hen egg white lysozyme (14.4 kDa). After low‐speed centrifugation of the clarified lysate and of the washed pellet homogenate (see , steps 5 and 7), the supernatants will be cloudy (lane f) and the pellets usually consist of two layers (see Fig. 6.1.5). The bottom layer is inclusion body protein plus unbroken cells (lanes b and c) and the top layer consists of outer membrane and peptidoglycan fragments (lanes d and e). The outer membrane proteins OmpA (35 kDa) and OmpF/C (38 kDa) are indicated by Ω; and o, respectively. After the washing steps, the growth hormone (marked β, 21 kDa) is the major constituent (lanes h and i) together, in this example, with another plasmid‐encoded protein, namely kanamycin phosphotransferase (marked α, 30.8 kDa), the product of the gene conferring resistance to the antibiotic kanamycin.
  •   FigureFigure 6.3.2 Gel filtration of an extract containing HIV‐1 protease, using Superdex 200 in 4 M guanidine⋅HCl. Column dimensions, 6 × 60 cm; buffer, 50 mM Tris⋅Cl (pH 7.5)/4 mM guanidine⋅Cl/2 mM DTT; flow rate, 5 ml/min (300 ml/hr). The sample has a mass of 10 kDa. Protein fractions 66 to 72 (pool P) were further purified under the same conditions using a Superdex 75 matrix. The inset shows SDS‐PAGE analysis of selected fractions. The protein standard markers (lane S) correspond to mass values of 66.2, 45, 30, 21.5, and 14.4 kDa, respectively (migration order top to bottom).
  •   FigureFigure 6.3.3 Superdex 200 chromatography in guanidine⋅HCl of SIV gp4127‐149. SDS‐PAGE of the numbered fractions is shown in the first inset (upper left); lane “a” contains molecular weight standards (bottom to top: 6.5, 14.4, 21.5, 31, 45, 66.2 kDa), and the purified protein migrates close to the 14.4 kDa standard. Lane “b” represents starting material loaded to column corresponding to guanidine⋅HCl‐extracted inclusion bodies. Protein in the main peak (fractions 5 to 7) marked with arrow was used for protein folding after removal of guanidine⋅HCl by reversed‐phase chromatography. The second inset (upper middle) refers to protein expression in minimal medium; lane A contains the same molecular weight standards as lane “a” in the first inset; lanes B and C correspond to insoluble protein and purified protein, respectively. Protein is labeled with 15N and 13C for NMR analysis. A summary of the protein purification is indicated in the right‐hand part of the figure. Adapted from Wingfield et al. ().


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