Preparation of Soluble Proteins from Escherichia coli

Paul T. Wingfield1

1 NIAMD/NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 6.2
DOI:  10.1002/0471140864.ps0602s78
Online Posting Date:  November, 2014
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Abstract

Purification of human IL‐1β is used in this unit as an example of the preparation of a soluble protein from E. coli. Bacteria containing IL‐1β are lysed, and IL‐1 β in the resulting supernatant is purified by anion‐exchange chromatography, salt precipitation, and cation‐exchange chromatography, and then concentrated. Finally, the IL‐1 β protein is applied to a gel‐filtration column to separate it from remaining higher‐ and lower‐molecular‐weight contaminants, the purified protein is stored frozen or is lyophilized. The purification protocol described is typical for a protein that is expressed in fairly high abundance (i.e., >5% total protein) and accumulates in a soluble state. In addition, the purification procedure serves as an example of how to use classical protein purifications methods, which may also be used in conjunction with the affinity‐based methods now more commonly used. © 2014 by John Wiley & Sons, Inc.

Keywords: recombinant protein; protein purification; interleukin‐1beta

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Purification of a Protein Expressed in Escherichia Coli in a Soluble State: Interleukin 1β
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Purification of a Protein Expressed in Escherichia Coli in a Soluble State: Interleukin 1β

  Materials
  • DEAE Sepharose CL‐4B resin (GE Healthcare Life Sciences)
  • Anion‐exchange buffer (see recipe)
  • 0.26% (w/v) sodium hypochlorite/70% ethanol or 5% (v/v) bleach (e.g., Clorox)/70% ethanol
  • E. coli cells (∼50 g wet weight) from fermentation (unit 5.3) containing IL‐1β
  • Lysis buffer (see recipe)
  • Bovine pancreas DNase I and RNase A (Worthington Biochemical; optional, for reducing solution viscosity)
  • 2 N sodium hydroxide
  • Ammonium sulfate, ground with mortar and pestle
  • Cation‐exchange buffer (see recipe)
  • CM Sepharose CL‐4B (GE Healthcare
  • Cation‐exchange buffer/250 mM NaCl (see recipe)
  • Tris base
  • Gel‐filtration buffer (see recipe)
  • Ultrogel AcA54 gel‐permeation resin (Pall Corporation; Sigma‐Aldrich)
  • Lyophilization buffer (see recipe; optional)
  • 2‐ or 3‐liter sintered glass funnel with fritted disc (coarse porosity) and 5‐liter filter flask
  • Chromatography columns (preferably glass) with adjustable flow adapters: one (or optionally two) 5 × 50 cm and one 2.5 × 100 cm (GE Healthcare, Amicon, or equivalent)
  • RK50 packing reservoir (GE Healthcare)
  • Peristaltic pump, UV monitor, and fraction collector (GE Healthcare; check online for other suppliers of equivalent items)
  • 16 × 150–mm culture tubes
  • 40‐ml French pressure cell and rapid‐fill kit (Thermo Fisher Scientific—see Commentary section on breaking cells)
  • French laboratory press (Thermo Fisher Scientific)
  • 1‐liter Waring commercial blender.
  • 250, 500, and 1000‐ml stainless steel beakers
  • Ice bucket, ∼4 liter
  • Tissue‐grinder homogenizer (Polytron Model PT 10/35, Brinkmann: various suppliers)
  • Ultrasonic homogenizer, ≥400 W, with sound enclosure (Branson or equivalent)
  • Preparative centrifuge: Beckman J2‐21M or Avanti J series (Beckman Coulter)
  • Rotors for preparative centrifuge: Beckman JA‐14 (capacity 6 × 250 ml) or JA‐20 (capacity 8 × 50 ml)
  • Ultracentrifuge: Beckman Optima XL‐90 or Optima L‐90 k (check Beckman Coulter Web site for other suitable models)
  • Rotors for ultracentrifuge: Beckman 45Ti (capacity 6 × 100 ml) or 35Ti (capacity 6 × 94 ml)
  • Conductivity meter (Radiometer Analytical)
  • Spectra/Por 1 dialysis tubing (Spectrum Labs)
  • Gradient maker: Model GM‐2000 (1000 ml per side with side outlet; CBS Scientific); smaller‐capacity gradient makers are also available (GE Healthcare and others)
  • 200‐ or 400‐ml stirred ultrafiltration cell and PM10 or YM3 Ultracel Amicon ultrafiltation discs (EMD Millipore)
  • Millex‐GV 0.22‐μm‐pore‐size filter units (EMD Millipore)
  • 10‐ or 20‐ml syringes
  • Additional materials and equipment for SDS‐PAGE (unit 10.1) and dialysis ( appendix 3B)
NOTE: All protocol steps are carried at 4°C unless otherwise stated. Forces for centrifugation steps refer to the maximum × g (i.e., centrifugal force at the bottom of the tubes).
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Figures

Videos

Literature Cited

Literature Cited
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  Wingfield, P.T., Graber, P., Rose, K., Simona, M.G., and Hughes, G.J. 1987. Chromatofocusing on N‐terminally processed forms of proteins. J. Chromatogr. 387:291‐300.
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Key References
  Burgess, R.R, and Deutscher, M.P. (eds.) 2009. Guide to protein purification. In Methods in Enzymology, vol. 463. Academic Press, San Diego.
  Good coverage of updated protein biochemistry and purification methods.
  Janson, J.‐C. and Ryden, L., (eds.) 1998. Protein Purification: Principles, High Resolution Methods and Applications, 2nd ed. John Wiley & Sons, Hoboken, N.J.
  Useful reference on protein purification.
  Scopes, R.K. 1994. See above.
  Emphasizes first principles.
  Simpson, R.J. (ed.) 2004. Purifying Proteins for Proteomics: A Laboratory Manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.
  Well‐illustrated coverage of modern protein purification methods.
  Wingfield et al., 1986. See above.
  The original publication on which the Basic Protocol is based.
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