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Preparation of Soluble Proteins from Escherichia coli
Publication Name:
Current Protocols in Protein Science
Unit Number:
Unit 6.2
DOI:
10.1002/0471140864.ps0602s41
Online Posting Date:
September, 2005 Abstract
Purification of human IL-1 is used in this unit as an example of the preparation of soluble proteins from E. coli. Bacteria containing IL-1 are lysed, and IL-1 in the resulting supernatant is purified by anion-exchange chromatography, salt precipitation and cation-exchange chromatography, and then concentrated. Finally, the IL-1 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.
Materials
Basic Protocol: Purification of a Protein Expressed in Escherichia coli in a Soluble State: Interleukin 1
Materials
- DEAE Sepharose CL-4B resin (Amersham Biosciences)
- 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
- Bovine pancreas DNase I and RNase A (Worthington Biochemical; optional, for reducing solution viscosity)
- Ammonium sulfate, ground with mortar and pestle
- CM Sepharose CL-4B (Amersham Biosciences)
- Tris base
- Ultrogel AcA54 gel-permeation resin (BioSepra; Ciphergen)
- 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 (Amersham Biosciences, Amicon, or equivalent)
- RK50 packing reservoir (Amersham Biosciences)
- Peristaltic pump, UV monitor, and fraction collector (Amersham Biosciences or equivalent)
- 16 × 150–mm culture tubes
- 40-ml French pressure cell and rapid-fill kit (Thermo Electron Corp.)
- French laboratory press (Thermo Electron Corp.)
- 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)
- Ultrasonic homogenizer, ³400 W, with sound enclosure (Branson or equivalent)
- Preparative centrifuge: Beckman J2-21M or Avanti J series
- 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
- 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 (Amersham Biosciences)
- 200- or 400-ml stirred ultrafiltration cell and PM10 or YM3 Ultracel Amicon ultrafiltation discs (Millipore)
- Millex-GV 0.22-µm-pore-size filter units (Millipore)
- 10- or 20-ml syringe
- Additional materials and equipment for SDS-PAGE (unit
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
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Figure 6.2.1Cell component distribution and typical expression levels obtained in E. coli. The asterisks (*) refer to purification yields of proteins expressed in E. coli in the soluble or insoluble states. -
Figure 6.2.2Scheme for purifying human interleukin-1. -
Figure 6.2.3Purification of IL-1. (A) SDS-PAGE analysis of samples at various stages. Analysis was conducted on a gel of dimensions 12 cm × 16 cm × 1.5 mm. Lane a, purified protein (100 µg loaded); lane b, purified protein (10 µg loaded); lane d, CM Sepharose pool (80%); lane e, DEAE Sepharose pool after ammonium sulfate fractionation (56%); lane f, high-speed supernatant (starting material for DEAE Sepharose column; 13.5%); lane g, cell lysate (12.0%). The percentages refer to specific IL-1 contents of the fractions determined by densitomeric scanning of the Coomassie blue–stained gel lanes. Lanes c and h contain the following protein standards (low-range standards supplied by 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 white lysozyme (14.4 kDa). (B) Analysis of results from gel filtration on Ultrogel AcA54. The excluded volume (V0 ) and the fully included volume (Vi ) are indicated. Inset, analytical rechromatography of the protein from the pooled fractions (indicated P in larger chromatogram). -
Figure 6.2.4Common methods used to purify soluble recombinant proteins. Abbreviations: IMAC, immobilized metal affinity chromatography; HIC, hydrophobic interaction chromatography. -
Figure 6.2.5Checklist of issues to consider when planning a purification strategy. -
Figure 6.2.6Initial protein purification methods typically involve centrifugation (the specific conditions vary according to culture scale and location of expressed protein), (NH4 )2 SO4 precipitation, or polyethylene glycol (PEG) precipitation. -
Figure 6.2.7Purification of the HIV-1 protein Nef and the anti-HIV protein MAP30. -
Figure 6.2.8Purification of the DNA-binding protein Ner of bacteriophage Mu. This basic (pI ~ 10) 9-kDa protein regulates phage lysogenic and lytic activity. -
Figure 6.2.9Issues to consider for ion exchange chromatography. -
Figure 6.2.10Chromatofocusing of IL-1. (A) Theoretical titration curve of IL-1 with N-terminal methionine (M+ ) and N-terminal alanine (M– ). (B) Chromatofocusing of a mixture of IL-1 M+ and IL-1 M– . Chromatography was performed using a Mono HR5/20 column (Amersham Bioscience) as described by Wingfield et al. (
Literature Cited
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| Choi, J.H. and Lee, S.Y. 2004. Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl. Microbiol. Biotechnol. 64:625-635. | |
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| Clore, G.M., Wingfield, P.T., and Gronenborn, A.M. 1991. High-resolution structure of interleukin 1 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. | |
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| 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 mutagensis 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-1 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-1 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-1. 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 1. EMBO J. 7:339-343. | |
| Saito, S. and Tsuchiya, T. 1984. Characteristics of n-octyl -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-1 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-1. 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-1 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-1. 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 Reference | |
| Deutscher, M.P., (ed.) 1990. Guide to Protein Purification. In Methods in Enzymology, vol. 182. Academic Press, San Diego. | |
| Good coverage of classical protein 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 | |
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