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

  • 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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Literature Cited

Literature Cited
  Allet, B., Payton, M., Mattalino, R.J., Turcatti, G., Gronenborn, A.M., Clore, G.M., and Wingfield, P.T. 1988. The purification and characterization of the bacteriophage Mu DNA‐binding protein Ner. Gene 65:259‐268.
  Bass, S. and Yang, M. 1997. Expressing cloned genes in Escherichia coli. In Protein Function: A Practical Approach, 2nd ed. (T.E. Creighton, ed) pp. 29‐55. IRL Press, Oxford.
  Burgess, R.R. and Jendrisak, J.J. 1975. A procedure for the rapid, large‐scale purification of E. coli DNA‐dependent RNA polymerase involving polymin P precipitation and DNA‐cellulose chromatography. J. Biol. Chem. 14:4634‐4638.
  Choi, J.H. and Lee, S.Y. 2004. Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl. Microbiol. Biotechnol. 64:625‐635.
  Chrunyk, B.A., Evans, J., Lillquist, J., Young, P., and Wetzel, R. 1993. Inclusion body formation and protein stability in sequence variants of Interleukin‐1β. J. Biol. Chem. 268:18053‐18061.
  Clore, G.M., Wingfield, P.T., and Gronenborn, A.M. 1991. High‐resolution structure of interleukin 1β in solution by three‐ and four‐dimensional nuclear magnetic resonance spectroscopy. Biochemistry 30:2315‐2323.
  Dinarello, C.A. 1989. Interleukin‐1 and its biologically related cytokines. Adv. Immunol. 44:153‐205.
  Gery, I. and Schmidt, J.A. 1985. Human interleukin 1. Methods Enzymol. 116:456‐467.
  Hlodan, R. and Hartl, F.U. 1994. How the protein folds in the cell. In Mechanisms of Protein Folding (R.H. Pain, ed.) pp. 194‐228. IRL Press, Oxford.
  Hopkins, T.R. 1991. Physical and chemical cell disruption for the recovery of intracellular proteins. In Purification and Analysis of Recombinant Proteins (R. Seetharam and S.K. Sharma, eds.) pp. 57‐83. Marcel Dekker, New York.
  Ito, T. and Wagner, G. 2004. Using codon optimization, chaperone co‐expression, and rational mutagenesis for production and NMR assignments of human eIF2a. J. Biomol. NMR. 28:257‐267.
  Johnson, B.H. and Hecht, M.H. 1994. Recombinant proteins can be isolated from E. coli by repeated cycles of freezing and thawing. Bio/Technology 12:1357‐1360.
  Joseph‐Liauzun, E., Legoux, R., Guerveno, V., Marchese, E., and Ferra, P. 1990. Human recombinant interleukin‐1β isolated from E. coli by simple osmotic shock. Gene 86:291‐295.
  Kronheim, S.R., Cantrell, M.A., Deeley, M.C., March, C.J., Glackin, P.J., Anderson, D.M., Hemenway, T., Merriam, J.E., Cosman, D., and Hopp, T.P. 1986. Purification and characterization of human interleukin‐1 expressed in Escherichia coli. Bio/Technology 4:1078‐1082.
  Livi, G.P., Lillquist, J.S., Ferrara, A., Sathe, G.M., Simon, P.L., Meyers, C.A., Gorman, J.A., and Young, P.R. 1991. Secretion of N‐glycosylated interleukin‐1β in Saccharomyces cerevisiae using a leader peptide from Candida albicans. Effect of N‐linked glycosylation on biological activity. J. Biol. Chem. 266:15348‐15348.
  McMahan, C.J., Slack, J.L., Mosley, B., Cosman, D., Lupton, S.D., Brunton, L.L., Grubin, C.E., Wignall, J.M., Jenkins, N.A., Brannan, C.I., Copeland, N.G., Huebner, K., Croce, C.M., Cannizzarro, L.A., Benjamin, D., Dower, S.K., Spriggs, M.K., and Sims, J.E. 1991. A novel IL‐1 receptor, cloned from B cells by mammalian expression, is expressed in many cell types. EMBO J. 10:2821‐2832.
  Meyers, C.A., Johanson, K.O., Miles, L.M., McDevitt, P.J., Simon, P.L., Webb, R.L., Chen, M.‐J., Holskin, B.P., Lillquist, J.S., and Young, P.R. 1987. Purification and characterization of human recombinant interleukin‐1β. J. Biol. Chem. 262:11176‐11181.
  Nash, H.A., Robertson, C.A., Flamm, E., Weisberg, R.A., and Miller, H. 1987. Overproduction of Escherichia coli integration host factor, a protein with nonidentical subunits. J. Bacteriol. 169:4124‐4127.
  Neidhardt, F.C. 1987. Chemical composition of Escherichia coli. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (F.C. Neidhardt, J.L. Ingraham, K.B. Low, B. Magasanik, M. Schaechter, and H.E. Umbarger, eds.) pp. 3‐6. American Society for Microbiology, Washington, D.C.
  Priestle, J.P., Schar, H.‐P., and Grutter, M.G. 1988. Crystal structure of the cytokine interleukin 1β. EMBO J. 7:339‐343.
  Saito, S. and Tsuchiya, T. 1984. Characteristics of n‐octyl β‐D‐thioglucopyranoside, a new non‐ionic detergent useful for membrane biochemistry. Biochem. J. 222:829‐832.
  Schreuder, H., Tardif, C., Trump‐Kallmeyer, S., Soffientini, A., Sarubbi, E., Akeson, A., Bowlin, T., Yanofsky, S., and Barret, R.W. 1997. A new cytokine‐receptor binding mode revealed by the crystal structure of the IL‐1 receptor with an antagonist. Nature 386:194‐200.
  Scopes, R.K. 1994. Protein Purification: Principles and Practice, 3rd ed. Springer‐Verlag, New York and Heidelberg.
  Sherwood, R.F. 1992. Making bacterial extracts suitable for chromatography. Meth. Mol. Biol. 11:287‐305.
  Thornberry, N.A., Bull, H.G., Calaycay, J.R., Chapman, K.T., Howard, A.D., Kostura, M.J., Miller, D.K., Molineaux, S.M., Weidner, J.R., Aunins, J., Elliston, K.O., Ayala, J.M., Casano, F.J., Chin, J., Ding, G.J.‐F., Egger, L.A., Gaffney, E.P., Limjuco, G., Palyha, O.C., Raju, S.M., Rolando, A.M., Salley, J.P., Yamin, T.‐T., and Tocci, M.J. 1992. A novel heterodimeric cysteine protease is required for interleukin‐1β processing in monocytes. Nature 356:768‐774.
  Vigers, G.P.A., Anderson, L.J., Caffes, P., and Brandhuber, B.J. 1997. Crystal structure of the type‐1 interleukin‐1 receptor complexed with interleukin‐1β. Nature 386:190‐194.
  Wingfield, P., Payton, M., Tavernier, J., Barnes, M., Shaw, A., Rose, K., Simona, M.G., Demczuk, S., Williamson, K., and Dayer, J.M. 1986. Purification and characterization of human interleukin‐1β expressed in recombinant Escherichia coli. Eur. J. Biochem. 160:491‐497.
  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.
  Wood, W.I. 1976. Tables for the preparation of ammonium sulfate solutions. Anal. Biochem. 73:250‐257.
  Yem, A.W., Richard, K.A., Staite, N.D., and Deibel, M.R. 1988. Resolution and biological properties of three N‐terminal analogues of recombinant human interleukin‐1β. Lymphokine Res. 7:85‐92.
  Zimmerman, S.B. and Trach, S. 1991. Estimation of macromolecular concentrations and excluded volume effects in the cytoplasm of Escherichia coli. J. Mol. Biol. 222:599‐620.
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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