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Fermentation and Growth of Escherichia coli for Optimal Protein Production

Alain Bernard¹, Mark Payton¹

¹Glaxo Institute for Molecular Biology, Geneva, Switzerland

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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Unit Introduction
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
Bibliography
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 Alternate Protocol 1)
ECPM1 medium (see recipe)
85% (w/v) H₃PO₄
28% (w/v) NH₄OH
10 N NaOH

Fermentor (Fig. 5.3.1)
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. 5.3.2; 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

Additional Materials (also see Basic Protocol 1)

HCDF1 medium (see recipe)
HCDM1 medium (see recipe), containing 10 g/liter glycerol
O₂ gas, pure

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

Additional Materials (also see Basic Protocol 1)

Methionine auxotroph host strain: E. coli DL41 or B834 (Table 5.2.1)
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

Additional Materials (also see Basic Protocol 1)

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 ¹⁵NH₄Cl stock solution
D₂O
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

Figure 5.3.1

Schematic diagram of a bacterial fermentor, depicting major functions. Variations in the design can be made to suit specific requirements.

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Figure 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.

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Figure 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 Alternate Protocol 1. (C) Kinetics of cumulated CO₂ production. Mass spectrometry of the exhaust gas was performed as described by Todd (1989). Acetate formation was measured as described by Reiling et al. (1985).

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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.