Fermentation and Growth of Escherichia coli for Optimal Protein Production

Alain Bernard1, Mark Payton1

1 Glaxo Institute for Molecular Biology, Geneva, null
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.3
DOI:  10.1002/0471140864.ps0503s00
Online Posting Date:  May, 2001
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Abstract

Large‐scale production of recombinant proteins in Escherichia coli requires growth of cells in fermentors. This unit lists E. coli strains appropriate for use in fermentors, and also discusses important characteristics of fermentation equipment. Production of recombinant proteins in batch fermentations is described, as are variations of fermentation systems that enable continuous growth and protein production in high‐cell‐density, fed‐batch cultures and that permit labeling of recombinant proteins with heavy atom derivatives such as selenomethionine or with stable isotopes such as 2H, 13C, and 15N. Production of labeled proteins facilitates structural studies by X‐ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. The protocols in this unit are designed for expression systems directing intracellular or periplasmic localization of recombinant proteins; however, in the case of extracellular secretion of the desired protein, the culture medium itself, rather than pelleted cells, would be saved, concentrated, and subjected to purification processes. Fermentation experiments require careful monitoring of cell growth and assurance of preinoculation sterility, both which are described here.

     
 
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Table of Contents

  • Basic Protocol 1: Production of Recombinant Proteins in Batch Fermentations
  • Alternate Protocol 1: Protein Production in High‐Cell‐Density Processes: Fed‐Batch Methods
  • Alternate Protocol 2: In Vivo Labeling of Recombinant Proteins with Heavy Metal Derivatives
  • Alternate Protocol 3: Stable Isotopic Labeling of Recombinant Proteins
  • Support Protocol 1: Growth Monitoring
  • Support Protocol 2: Sterility Checking
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Production of Recombinant Proteins in Batch Fermentations

  Materials
  • Transformed E. coli host strain expressing the protein of interest (unit 5.2)
  • LB medium with antibiotic (unit 5.2)
  • Antifoam: polypropylene glycol (PPG) 2000 (pure; store indefinitely at room temperature)
  • Nutrient solution (optional, for fed‐batch methods; see protocol 2)
  • ECPM1 medium (see recipe)
  • 85% (w/v) H 3PO 4
  • 28% (w/v) NH 4OH
  • 10 N NaOH
  • Fermentor (Fig. )
  • 5‐ml and 50‐ml sterile syringes and 1 × 40–mm needles
  • Centrifuge, with capacity up to 6 liters per run (for 1‐ to 10‐liter scale; e.g., Sorvall RC3B, equipped with H6000A rotor) or continuous feed centrifuge (for scale >10 liters; Fig. ; e.g., Sharples AS16)
  • Heat‐sealable plastic bags and heat‐sealing apparatus
  • Additional reagents and equipment for inducing protein expression and for preparing samples for polyacrylamide gel analysis (unit 5.2)

Alternate Protocol 1: Protein Production in High‐Cell‐Density Processes: Fed‐Batch Methods

  • HCDF1 medium (see recipe)
  • HCDM1 medium (see recipe), containing 10 g/liter glycerol
  • O 2 gas, pure

Alternate Protocol 2: In Vivo Labeling of Recombinant Proteins with Heavy Metal Derivatives

  • Methionine auxotroph host strain: E. coli DL41 or B834 (Table 97.80.4711)
  • Expression vector containing gene for the protein of interest
  • DLM medium (see recipe)
  • Complete rich medium (e.g., LB medium; unit 5.2)
  • Additional reagents and materials for electroporation and testing expression of transformants (unit 5.2)

Alternate Protocol 3: Stable Isotopic Labeling of Recombinant Proteins

  • Transformed E. coli host strain expressing the protein of interest (unit 5.2)
  • HCDM1 medium (see recipe)
  • 50% (w/v) [13C] glucose
  • 100 g/liter 15NH 4Cl stock solution
  • D 2O
  • Base for pH correction: 2 N NaOH (for 15N label)

Support Protocol 1: Growth Monitoring

  Materials
  • Test sample (e.g., E. coli inoculum or fermentation medium)
  • Culture medium (optional, for diluting visibly turbid cultures)
  • Spectrophotometry cuvettes (e.g., 1‐cm light path)
  • Fixed‐wavelength spectrophotometer (e.g., 600 or 660 nm)
  • Count slide or hemacytometer
  • Phase‐contrast microscope (400×)

Support Protocol 2: Sterility Checking

  Materials
  • Test sample (e.g., fermentation medium)
  • LB plates with and without antibiotic (unit 5.2)
  • 0.45‐µm membrane (Millipore, type HA or HC)
  • Sterile filter holder
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Figures

  •   FigureFigure 5.3.1 Schematic diagram of a bacterial fermentor, depicting major functions. Variations in the design can be made to suit specific requirements.
  •   FigureFigure 5.3.2 Model AS16 continuous feed tubular bowl centrifuge (Alfa Laval). Other models (disc stack, with pellet ejection) are suitable for pilot‐scale operations. Broth is fed by pressure at the bottom of the bowl, and cells sediment against a liner inside the bowl. Cell‐free supernatant flows from the top of the bowl. The capacity is ∼6 liters. A cooling jacket is provided to avoid high temperatures.
  •   FigureFigure 5.3.3 High‐cell‐density growth. (A) Kinetics of growth (OD600) and acetate production. (B) Feed rate profile. Note that the feed rate was adjusted according to growth rate as described in . (C) Kinetics of cumulated CO2 production. Mass spectrometry of the exhaust gas was performed as described by Todd (). Acetate formation was measured as described by Reiling et al. ().

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Literature Cited

Literature Cited
   Bailey, F.J., Warf, R.T., and Maigetter, R.Z. 1990. Harvesting recombinant microbial cells using crossflow filtration. Enzyme Microb. Technol. 12:647‐652.
   Bauer, S. and White, M.D. 1976. Pilot scale exponential growth of Escherichia coli to high cell concentration with temperature variation. Biotech. Bioeng. 18:839‐846.
   Clore, G.M. and Gronenborn, A.M. 1994. Multidimensional heteronuclear nuclear magnetic resonance of proteins. Methods Enzymol. 239:349‐363.
   Dusseljee, P.J.B. and Feijen, J. 1990. Instrumentation and control. In Fermentation, A Practical Approach. (B. McNeil and L.M. Harvey, eds.) pp. 149‐171. IRL press, Oxford.
   Gram, H., Ramage, P., Memmert, K., Gamse, R., and Kocher, H.P. 1994. A novel approach for high level production of a recombinant human parathyroid hormone fragment in Escherichia coli. Bio/Technology 12:1017‐1023.
   Halling, P.J. 1990. pH, dissolved oxygen and related sensors. In Fermentation, A Practical Approach. (B. McNeil and L.M. Harvey, eds.) pp. 131‐147. IRL press, Oxford.
   Knorre, W.A., Deckwer, W.D., Korz, D., Pohl, H.D., Riesenberg, D., and Ross, A. 1991. High cell density fermentation of recombinant Escherichia coli with computer controlled optimal growth rate. Ann. N.Y. Acad. Sci. 646:300‐306.
   Lech, K. and Brent, R. 1994a. Growth in liquid media. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 1.2.1‐1.2.2. John Wiley & Sons, New York.
   Lech, K. and Brent, R. 1994b. Growth on solid media. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 1.3.1‐1.3.5. John Wiley & Sons, New York.
   Mori, H., Yano, T., Kobayashi, T., and Shimizu, S. 1979. High density cultivation of biomass in fed‐batch system with DO‐stat. J. Chem. Eng. 12:313‐319.
   Reiling, H.E., Laurila, H., and Fiechter, A. 1985. Mass culture of Escherichia coli: Medium development for low and high density cultivation of Escherichia coli B/r in minimal and complex media. J. Biotech. 2:191‐206.
   Sinclair, C.G. and Cantero, D. 1990. Fermentation modelling. In Fermentation, A Practical Approach. (B. McNeil and L.M. Harvey, eds) pp. 65‐85. IRL press, Oxford.
   Schügerl, K., Brandes, L., Wu, X., Bode, J., Ree, J.I., Brandt, J., and Hitzmann, B. 1993. Monitoring and control of recombinant protein production. Anal. Chim. Acta 279:3‐16.
   Strandberg, L., Koehler, K., and Enfors, S.O. 1991. Large scale fermentation and purification of a recombinant protein from Escherichia coli. Process Biochem. 26:225‐234.
   Todd, D. 1989. Mass spectrometry of fermentation emissions. BioTechnology 7:1182‐1183.
   Yang, X.M. 1992. Optimization of a cultivation process for recombinant protein production by Escherichia coli. J. Biotechnol. 23:271‐289.
   Yang, W., Hendrickson, W.A., Kalman, E.T., and Crouch, R.J. 1990. Expression, purification and characterization of natural and selenomethionyl recombinant ribonuclease H from Escherichia coli. J. Biol. Chem. 265:13553‐13559.
   Yee, L. and Blanch, H.W. 1992. Recombinant protein expression in high cell density fed‐batch cultures of Escherichia coli. Bio/Technology 10:1550‐1556.
Key Reference
  Fermentation, A Practical Approach. 1990. (B. McNeil and L.M. Harvey, eds.) IRL press, Oxford.
  Covers the complete fermentation process cycle, illustrating many diagrams for equipment and giving essential background theoretical information.
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