Fermentation and Growth of Escherichia coli for Optimal Protein Production

Large-scale production of recombinant proteins in Escherichia coli requires growth of cells in fermentors. Fermentation involves the transformation of simple organic compounds by the enzymatic machinery of E. coli, in this case to produce proteins. There are many choices of bacterial strain, vector, expression system, and protein localization and purification strategy that must be set before proceeding to batch fermentation. UNIT 5.1 gives an overview of protein expression in E. coli, and UNIT 5.2 outlines procedures for transforming cells and inducing synthesis of foreign proteins in various expression systems on a small scale in shaker flasks. Table 5.2.1 lists E. coli strains appropriate for use in fermentors. The Commentary of this unit discusses important characteristics of fermentation equipment. Starting with a strain of E. coli that has been transformed to produce the desired protein (UNIT 5.2) this unit focuses on production of recombinant proteins in batch fermentations (Basic Protocol 1). Alternate protocols introduce variations of fermentation systems that enable continuous growth and protein production in high-cell-density, fed-batch cultures (Alternate Protocol 1) and that permit labeling of recombinant proteins with heavy atom derivatives such as selenomethionine (Alternate Protocol 2) or with stable isotopes such as 2H, 13C, and 15N (Alternate Protocol 3). 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, which are described in Support Protocols 1 and 2, respectively. It must be kept in mind, however, that the outcome of fermentation experiments, even if derived from successful small-scale shaker flask cultures (UNIT 5.2), may be negatively affected by unexpected toxicity of the target protein in fermentation systems. PRODUCTION OF RECOMBINANT PROTEINS IN BATCH FERMENTATIONS This protocol describes the basic sequence of operations, their timing, and the key issues associated with batch fermentations. Simple batch fermentations involve growth of fermenting organisms in a fixed amount of medium, without additional nutrients. The fermentor is set up, filled with fermentation medium (but not including heat-sensitive ingredients), and sterilized. Filter-sterilized heat-sensitive ingredients are then added, and environmental parameters are set to the desired values (using acid and base to adjust pH) before final preinoculation samples are taken to check sterility (Support Protocol 2). An inoculum prepared from a fresh overnight culture of the transformed E. coli strain is introduced into the fermentor and the cells allowed to grow. Synthesis of the recombinant protein following induction of cells is monitored, and cells are harvested by centrifugation using a batch pot or continuous feed centrifuge, depending on the volume of medium processed. Special fermentation equipment is required. Several commercial manufacturers produce fermentation equipment (Table 5.3.1) for laboratory use (1 to 20 liters) or small pilot-scale experiments (50 to 200 liters). For recombinant E. coli processes, users should pay particular attention to heat and oxygen transfer, process monitoring and control (particuContributed by Alain Bernard and Mark Payton
Current Protocols in Protein Science (1995) 5.3.1-5.3.18 Copyright 2000 by John Wiley & Sons, Inc.

UNIT 5.3

BASIC PROTOCOL 1

Production of Recombinant Proteins

5.3.1
CPPS

Table 5.3.1

Fermentation Equipment Suppliers

Suppliera B. Braun Bioengineering

Vessel size (liters) 2 to 50,000 2.4 to 50000

Control DDCb, Computer supervision Analog, DDC, Computer supervision

Comments Microprocessor-based instrumentation Special vessel designs (airlifts, fluidized beds, membrane dialysis) Complete plants Very high oxygen transfer Very high oxygen transfer Windows-based control package Microprocessor-based instrumentation Complete plants

Inceltech New Brunswick Scientific Co. LSL Biolafitte New MBR

Analog, DDC, Computer supervision 1 to 15,000 Analog, DDC, Computer supervision 1 to 50,000 DDC, Computer supervision 3.5 to 100,000 Analog, DDC, Computer supervision

2.5 to 800

aFor complete listing of suppliers addresses, see SUPPLIERS APPENDIX. bDDC, direct digital control

larly if high-cell-density processes are needed; Alternate Protocol 1), and ease of maintenance and servicing. Utilities such as air, water, steam, and electricity are needed in defined qualities and quantities. Consult the fermentor manufacturer for specific requirements. 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 40mm 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) Prepare the inoculum and fermentor 1. Prepare an overnight culture of the transformed E. coli host strain expressing the protein of interest by inoculating a suitable amount of LB medium, supplemented with the appropriate antibiotic to maintain plasmid-carrying cells, with a single colony picked from an LB/antibiotic plate or retrieved from a vial of frozen cells (see UNIT 5.2). The next morning, read the OD600 against water as a blank to verify that the

Fermentation and Growth of E. coli for Optimal Protein Production

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exhaust air

exhaust air filter

reflux cooler drain cold water inoculum antifoam base acid top

flow meter air

air inlet filter

sparger
reactor vessel

sample harvest

jacket

cold water
stirrer agitator

drain steam drain

mechanical seal

motor
electrically controlled valves

manual valve

circulating pump for water jacket

Figure 5.3.1 Schematic diagram of a bacterial fermentor, depicting major functions. Variations in the design can be made to suit specific requirements.

culture has reached stationary phase (i.e., OD>1.5, see Support Protocol 1). Use the overnight culture to inoculate the fermentor.
The size of the inoculum can be varied between 1% and 10% of the intended fermentation volume. For a 10-liter fermentor, the required inoculum of 100 to 1000 ml can be conveniently prepared in 500-ml to 5-liter shaker flasks (a maximum ratio of liquid/flask volume of 0.3 ensures that the culture is well aerated). Fermentors of sizes above 10 liters can be inoculated with several shaker flasks, representing 1% of the fermentor volume. The contents of all the inoculum flasks are pooled into a single large sterile container, fitted with the appropriate transfer line. The choice of antibiotic is dictated by the transformation vector, and the incubation temperature depends on the expression system (use 30C for pL-based systems and 37C for others). The inoculum is needed in step 15 after the fermentor has been loaded with fermentation medium and sterilized.

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motor

cooling water out

supernatant

cooling water in

feed

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.

2. Empty the reactor vessel of the fermentor and inspect it for residual biological material on the inner wall, agitator shaft baffles, or other parts.
Between fermentation processes, the fermentor is usually kept partially filled with water.

3. Calibrate sensors: pH and pO2 probes, load cells, and pressure gauges according to manufacturers instructions.
Although calibration is not necessary for all sensors and every run, it should be performed on a regular basis (every 10 runs or so). The pH and pO2 probes, however, should be recalibrated before each run because of the possible drift of the signal with time or fouling of the probe membrane. The calibration of the pO2 probe done here is a rough determination of whether the probe is responding to changes of dissolved O2. The precise calibration for pO2 is performed at step 10, after the probe is in the sterilized fermentation medium, in an environment closest to the one of the process.

4. Prepare accessory lines, transfer vessels, and ports, including septums and addition lines for inoculum, acid, base, antifoam, nutrients, and inducers. Sterilize by autoclaving. Also sterilize antifoam and, if applicable, nutrient solution, if possible while linked to the respective lines.
Some nutrient solutions (e.g., for fed-batch processes) contain heat-sensitive components and should be sterilized according to the recipe (see Reagents and Solutions).

Fermentation and Growth of E. coli for Optimal Protein Production

5. Prepare ECPM1 medium by mixing all heat-sterilizable ingredients with water to 80% of the final volume. Pour into fermentor.
For fermentation volumes exceeding 5 liters, dissolve all ingredients as a concentrate (at 100 the final required concentration) and pour into the fermentor.

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6. Adjust presterilization volume by adding water directly to the reactor vessel.

The presterilization volume should be calculated from the final culture volume (which should not exceed 34 of the total fermentor volume) by deducting volumes of inoculum, heat-sensitive ingredients and antibiotic to be added and water that evaporates during sterilization. The addition of water resulting from steam condensation during sterilization must also be taken into account. The amounts of liquid loss or gain should not exceed 10% of the final culture volume and is best empirically determined by measuring accurately the liquid level pre- and post-sterilization in a mock trial sterilization with water. Culture volume does vary during the process, due to mass cell accumulation, sampling, additions of inducers and for pH and foam correction, but these phenomena usually result in a net volume variation that is negligible (except for fed-batch processes).

Sterilize the fermentor and medium 7. Start sterilization cycle. For autoclavable glass transfer vessels (usually 1 to 5 liters), disconnect all cables and tubings from the fermentor; allow the inside of the chamber to be ventilated through one or two lines (protected with air filters) that communicate with the reactor headspace. For transfer vessels that can be sterilized in situ, engage the automatic sequence controller.
It is best to insert the temperature sensor of the autoclave into the temperature port of the reactor, if possible. This ensures that the autoclave control system will sense the exact temperature of the broth inside the reactor. For in situ sterilizable vessels, most fermentors are fitted with an automatic sequence controller that initiates and controls the whole sterilization cycle with predetermined parameters.

8. Sterilize at 121C for 30 min.

If cooling proceeds too quickly, an internal vacuum may form that will open the way to contamination. A cooling rate of 40C/min is acceptableif it is higher than this, restrict the cooling water flux.

9. As soon as the temperature reaches the range of 102 to 108C, apply headspace pressure to the reactor with sterile air (0.05 to 0.1 bar). Start water flow through the reflux cooler to condense exhaust gases.
Applying headspace pressure is important to avoid an internal vacuum and to create a barrier to any potential external contaminant. It is also good practice, once pressure has stabilized, to set a small air flow through the headspace (0.2 liters air/culture/min) to dry the exhaust gas filter.

Conduct final preparations for inoculation 10. For pH correction, pour 85% H3PO4 into one transfer vessel (acid) and 28% NH4OH into the other (base) under a biological hood. Connect acid, base, and antifoam lines aseptically to the fermentor. Once the temperature is 40C, proceed to step 11.
Aseptic connections are easily made by using a sterile needle to puncture a sterile septum port. Use flame sterilization and/or a 70% ethanol spray to ensure sterility. Some connectors can be sterilized again by steam after the connection is completed.

11. Add heat-sensitive ECPM1 medium ingredients (the 1 M MgCl26H2O previously sterilized by 0.22-m filtration; see Reagents and Solutions) with a sterile syringe by puncturing a septum port with the needle. 12. Calibrate pO2 sensor by sparging air at 0.5 vol gas/vol culture/min with vigorous agitation (500 rpm) and adjusting the 100% saturation point.
The zero point of the sensor can be calibrated by sparging N2 gas instead of air, or with a sensor simulator, but this point should not need frequent recalibrationafter every 10 sterilization cycles should suffice.

Production of Recombinant Proteins

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13. Adjust all environmental parameters to the desired set points.

Typical parameter settings are: temperature, 30 or 37C; pH, 7.0; agitation, 300 rpm; air flow, 1 vol/vol culture/min; pO2, 100% air saturation.

14. Withdraw a small sample aseptically and check for sterility (see Support Protocol 2).
A sterile fermentor can be prepared 2 to 3 days in advance. If this is done, it is best to recheck the sterility of a fresh sample taken just before inoculation. Because the results of this sterility check are not known for another 12 to 16 hr, the procedure is initiated and continued. In case of a contamination revealed after inoculation, the batch should be considered suspect and/or discarded. A massive contamination (which could occur if the fermentor has been prepared more than 24 hr in advance) will be obvious from the evolution of the pH and/or pO2 and turbidity of the sample. If any contamination is detected before inoculation, the entire run should be cancelled and the fermentor cleaned (see step 24).

Inoculate the fermentor, growth cells, and induce protein expression 15. Aseptically connect inoculum line to fermentor septum port. Inoculate fermentor by transferring the inoculum (step 1) aseptically into the reactor vessel. Disconnect inoculum line.
A short spray of 70% ethanol on the septum and needle improves sterility. Inoculation is achieved either via an in-line peristaltic pump or by pressurizing the inoculum vessel with sterile air.

16. Monitor growth by measuring OD600 of samples taken at 1-hr intervals (see Support Protocol 1). When the target cell concentration is reached, proceed to induction.
Target cell concentration depends on the strain and expression system. As a general rule, and in the absence of any previous experience with a given strain, a target of OD 5 to 10 is applicable. It is possible, however, that the same process can also be run with induction at a later stage (OD 15 to 20), but this needs empirical determination.

17. Induce protein expression via temperature or chemical induction using one of the following methods, as appropriate. a. For the pL system: Perform a fast temperature shift (within a few minutes) from 30 to 42C and incubate 5 hr. b. For the trp promoter system: Add IAA to a final concentration of 25 mg/liter (see UNIT 5.2). Incubate to stationary phase (usually overnight). c. For the lac and tac promoter and T7 systems: Add IPTG to a final concentration of 0.5 to 1.0 mM (UNIT 5.2) and incubate 5 hr. 18. At 1-hr intervals, withdraw samples of the culture, monitor growth by OD600 measurements, and prepare them for polyacrylamide gel analysis, as described in UNIT 5.2, to monitor recombinant protein expression.
A band of the expected molecular weight should appear in lanes of induced samples, but not in those of uninduced or early-time-of-induction samples. Results of this analysis cannot be known before the projected harvesting time, so for the first experiment, the process should be run with the time lines indicated above. The best combination of high expression (judged by band intensity on the gel) and high cell density should dictate optimal harvesting timethese conditions can then be applied to repeat experiments. It it also important to monitor growth closely during the induction phase because some strains rapidly lyse. In case of cell lysis, proceed to harvesting.
Fermentation and Growth of E. coli for Optimal Protein Production

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Harvest the cells For small fermentors (1 to 10 liters): 19a. To harvest cells from small fermentors, pressurize the fermentor and transfer the reactor contents into an intermediate container. 20a. Distribute liquid culture, preferably under a biological hood, into centrifuge bottles. Record final culture volume in order to estimate final cell yield.
Aerosols should be minimized at all times. Use closed containers fitted with sterile filter vents.

21a. Centrifuge 15 min at 5000 g (4000 rpm in an H6000A rotor), 4C. 22a. Collect pellets from each centrifuge bottle and place together in a heat-sealable plastic bag. Proceed to step 23.
If the protein is secreted to the extracellular medium, collect the supernatant and concentrate by tangential flow ultrafiltration.

For large fermentors (>10 liters): 19b. For larger fermentors, prepare for cell harvest by removing all flexible tubing connections on the reactor, stopping pH and pO2 control loops, and setting the temperature set point at minimum. Set pressure set point to 0.8 bars and direct gas flow to the headspace of the reactor.
In Figure 5.3.2, the broth is transferred from the fermentor to the bottom of a continuous feed centrifuge by pressurizing the reactor through the headspace. Consult the manufacturers instructions for specific operating conditions. It is important to cool the culture liquid quickly to minimize proteolytic degradation.

20b. Start the continuous feed centrifuge and accelerate to an effective centrifugation force of 10,000 g. When bowl speed is stable, open connecting valves from the fermentor to the centrifuge. 21b. Set inlet flow rate to the centrifuge at 1 to 2 liters/min. Centrifuge at 4C until all the medium has been processed. 22b. Stop centrifuge, dismount the bowl, and collect the pelleted cells into a heat-sealable plastic bag.
If the purification process cannot handle the entire pellet volume, divide the material into smaller aliquots. If the protein is secreted to the extracellular medium, collect the supernatant and concentrate by tangential flow ultrafiltration.

Store the harvested cells and clean the fermentor 23. Retain a small amount of the pellet (50 to 100 mg) in a separate microcentrifuge tube for polyacrylamide gel analysis (step 18) to verify that centrifugation has not altered the target protein expression level. Close pellet-containing bags by heat sealing. Record total pellet weight and freeze at 20C.
Most recombinant proteins in the pelleted cells remain stable for months when stored at 20C. However, some membrane receptors (e.g., neurokinin, adrenergic, transmembrane receptors in general) are best preserved by snap freezing in dry ice/isopropanol and storing at 70C.

24. To clean the fermentor, fill it with water and adjust to pH 11.0 to 12.0 by adding 10 N NaOH. Set temperature to 50C and leave with vigorous agitation overnight.
Local biosafety rules may require sterilization of the reactor before cleaning. For sterili-

Production of Recombinant Proteins

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zation, fill the reactor with water and proceed to steps 7 and 8. If the fermentor is still not clean after this treatment, alternate alkali and acid (H3PO4) treatments.

25. Empty the reactor, rinse extensively with water, and sterilize as described in steps 7 and 8. Prepare the fermentor for another run, if desired.
Electrochemical sensors (pH, pO2, pCO2) should be removed and stored according to manufacturers instructions. The fermentor should then be filled with water, sterilized, and stored sterile until the next run. ALTERNATE PROTOCOL 1

PROTEIN PRODUCTION IN HIGH-CELL-DENSITY PROCESSES: FED-BATCH METHODS Fed-batch fermentations take advantage of the high-protein-synthesizing capacity of an exponentially growing culture while minimizing the buildup of toxic by-products by keeping growth carbon sourcelimited. This protocol describes a simple glycerol-supplementation method that is applicable to a wide range of culture conditions. Cells are first grown in a batch fermentation in a medium containing 10 g/liter glycerol. After the initial glycerol is consumed, the culture is supplemented with an increasing feed rate of concentrated medium containing 900 g/liter glycerol. Except for cell density, induction of protein synthesis and harvest are the same as for batch fermentation (see Basic Protocol 1). Additional Materials (also see Basic Protocol 1) HCDF1 medium (see recipe) HCDM1 medium (see recipe), containing 10 g/liter glycerol O2 gas, pure 1. Prepare an inoculum culture of the transformed E. coli host strain expressing the protein of interest, then set up the fermentor as for batch fermentation (see Basic Protocol 1, steps 1 to 4). 2. Prepare HCDF1 feed medium in a feed tank (transfer vessel) and set on a balance or a load cell to keep track of feed consumption. 3. Proceed to preparing the fermentation medium, sterilizing the medium and fermentor, and conducting the final preparations for inoculation for batch fermentation procedure (see Basic Protocol 1, steps 5 to 14), substituting HCDM1 batch medium containing 10 g/liter glycerol as the fermentation medium. 4. Inoculate the fermentor and grow cells (see Basic Protocol 1, steps 15 and 16). After the initial glycerol is consumed, start the feed of HCDF1 medium.
A sharp rise in pO2 is indicative of reduced oxygen consumption and thus of glycerol becoming limiting. Monitor growth during this phase to estimate maximum growth rate (see Sinclair and Cantero, 1990, for methodology).

5. Increase rate of addition of HCDF1 medium gradually and withdraw samples to follow the increase in OD600. Adjust rate of HCDF1 medium addition to the target feed rate. Monitor feed rates by measuring the mass of the feed tank over time.
The target feed rate can be estimated as F = Vf OD/(Y 0.4 0.9)
Fermentation and Growth of E. coli for Optimal Protein Production

where F is the feed rate (ml/hr), is the target growth rate (hr1), Vf is the fermentor volume (liters), OD is the optical density at 600 nm, Y is the yield of cell mass per unit carbon source (g/g), 0.4 represents the conversion factor from OD to cell mass (OD/g/liter), and 0.9 is the glycerol content in the feed solution (g/ml). An estimate of

5.3.8
Current Protocols in Protein Science

80% of the maximum growth rate (step 4) is a good starting value. An estimate of 0.3 is a good value for the yield, Y. When monitoring feed rates, make sure to take into account the density of the feed and the increase in fermentation volume with feeding. The target growth rate is dependent on each strain and each process. To avoid accumulation of toxic by-products (organic acids), the target growth rate should be set below 80% of the maximum growth rate observed during the batch phase.

6. Blend pure O2 gas into the air that is sparged into the reactor (either by solenoid valve pulses or by a motor-driven proportional valve) to make sure that pO2 is properly controlled (set point between 20% and 50%). 7. Once the target high biomass density is obtained (OD600 above 50), induce expression of the protein as in batch fermentation procedure (see Basic Protocol 1, step 17).
Final cell densities can be as high as 100 to 150 OD units. Induction by a chemical inducer can also be achieved by simply adding the inducer to the feed solution (gradual induction).

8. Proceed to monitoring expression and harvesting (see Basic Protocol 1, steps 18 to 23) while continuing to match the rate of HCDF1 supplementation with growth. IN VIVO LABELING OF RECOMBINANT PROTEINS WITH HEAVY METAL DERIVATIVES To maximize incorporation of selenomethionine in place of methionine in recombinant proteins to facilitate structural studies, it is preferable to use a methionine auxotroph host strain. The protocol is best suited for small fermentors (1 to 10 liters) because the medium change step requires sterile harvesting via a batch pot centrifuge. 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) Construct the expression strain 1. Transform methionine auxotroph host strain with expression vector containing the gene for the protein of interest (see UNIT 5.2).
For T7 expression systems, use E. coli B834 (with or without pLys plasmid) as a host (also see UNIT 5.2 Commentary and see Table 5.2.1). ALTERNATE PROTOCOL 2

2. Test expression of transformants in a shaker flask induction test (see UNIT 5.2). Set up the fermentation and induce protein expression 3. Prepare DLM medium (same volume as fermentor) and keep at room temperature. 4. Prepare fermentor with complete rich medium and inoculate with the recombinant strain (see Basic Protocol 1, steps 1 to 16). 5. Grow overnight in rich medium at 30C for pL strains, 37C for others.
Production of Recombinant Proteins

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Current Protocols in Protein Science

6. Withdraw sample and measure OD600. Harvest cells under sterile conditions by batch pot centrifugation (see Basic Protocol 1, steps 19a to 22a). Stop all control loops on the fermentor.
OD values should be in the range of 20 to 30 after overnight growth. Harvest only part of the culture. Calculate the volume to be harvested so that, once the cells are resuspended in the same volume of DLM labeling medium, the OD600 will be between 10 and 15.

7. Resuspend the pellet in DLM medium. Transfer to a sterile container and then back to the fermentor. Resume all control loops.
In practice, there will be several pellets, distributed in several centrifuge bottles. Pellets are individually resuspended, then suspensions are pooled into a single vessel to avoid having to transfer each separate bottle back into the fermentor. Remember that sterility is a must here, and handling one container is less risky than dealing with several.

8. Wait for 30 min until all environmental parameters are back to their set points and are stable before inducing expression (see Basic Protocol 1, step 17). Proceed to monitoring expression and harvesting cells (see Basic Protocol 1, steps 18 to 23).
ALTERNATE PROTOCOL 3

STABLE ISOTOPIC LABELING OF RECOMBINANT PROTEINS Uniform incorporation of stable isotopes in recombinant proteins is an alternative to selective labeling of a given amino acid (Alternate Protocol 2) to facilitate structural studies. The method requires the use of a semi-defined medium containing salts, trace elements, and yeast extract. Cells are supplied with [13C]glucose, 15NH4Cl, and/or D2O, and induced to synthesize recombinant protein labeled with 13C, 15N, and/or 2H. The three labels can be used individually or in combination. This protocol makes use of the fed-batch method (Alternate Protocol 1) but substitutes (labeled) glucose for glycerol as the carbon source and is run at much lower cell densities. 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) 1. Grow an inoculum culture of the transformed E. coli host strain expressing the protein of interest, then prepare fermentor and start the batch fermentation with HCDM1 medium (see Basic Protocol 1, steps 2 to 14), setting up 50% [13C] glucose as the nutrient solution (place feed tank on a balance or load cell).
In this protocol, the carbon source is labeled glucose instead of glycerol. For 2H labeling, use D2O instead of H2O in making up HCDM1 medium.

2. After medium sterilization add 10 g/l [13C]glucose and the first 5-ml portion of 100 g/liter 15NH4Cl stock solution per liter fermentation volume (using a sterile syringe; see Basic Protocol 1, step 12).
Glucose is added after sterilization to avoid partial destruction when heat sterilized with nitrogen-containing compounds present in the medium. To achieve the target 10 g/liter concentration, add 40 ml of the feed solution per liter of culture.

Fermentation and Growth of E. coli for Optimal Protein Production

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15

NH4Cl is added in three separate batches to avoid inhibition by growth by excess NH4Cl in the medium.

3. Inoculate fermentor (see Basic Protocol 1, step 15). Allow cells to grow. When the initial [13C]glucose is consumed, start feed with [13C]glucose.
A sharp rise in pO2 is indicative of glucose becoming limiting and thereby reducing oxygen consumption. To set initial and subsequent feed rates, use the equation in Alternate Protocol 1, step 5, substituting 0.5 for 0.9 as the glucose feed content (g/ml), using a target growth rate of 0.3/hr and an estimate of 0.4 for the yield Y.

4. Monitor growth by withdrawing samples periodically and measuring OD600. At an OD600 5, add second batch of 15NH4Cl and induce protein expression (see Basic Protocol 1, step 17).
In this protocol a high cell density is not desirable, and therefore no pure O2 should be necessary for pO2 control.

5. At 1 hr postinduction add the last batch of 15NH4Cl. 6. Proceed to monitoring protein expression and harvesting (see Basic Protocol 1, steps 18 to 23) while continuing to match the rate of glucose supplementation with growth. GROWTH MONITORING Bacterial growth can be monitored off-line by several different methods (Lech and Brent, 1994a). For fermentation process control, spectrophotometry is fast, reliable, and simple. Microscope counting is an alternative but is more cumbersome and time-consuming. On-line sensors also exist and have been used successfully in some cases (Schgerl et al., 1993). Most sensors, however, are sensitive to particulate matter, gas bubbles, fouling, and calibration drifts. 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) For spectrophotometry: 1a. Place test sample in spectrophotometry cuvette. Fill reference cuvette with water. 2a. Transfer cuvettes to a fixed-wavelength spectrophotometer and measure the optical density at 600 nm.
The level of absorbance at 600 nm will depend on the distance between the cuvette and the detector and will vary among instruments, often by a factor of 2. It is thus wise to calibrate each instrument by recording the OD600 of a culture that contains a known number of cells determined by some other method, such as observation on a count slide or titering for viable colonies (Lech and Brent, 1994b). For a culture grown in rich medium, a good rule of thumb is that each 1 OD unit is roughly equivalent to 109 cells/ml. OD readings above 0.4 cannot be accurately correlated with cell numbers and the sample should be diluted 10- or 100-fold with PBS to bring reading back into the 0.4 range.
Production of Recombinant Proteins

SUPPORT PROTOCOL 1

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For cell counting: 1b. Take a clean count slide (or hemacytometer) and cover it with a clean coverslip. Dip a 0.1- or 1-ml pipet into the test sample, allow a small drop of liquid to form on the end of the pipet, and touch it lightly to the surface of the slide at the periphery of the coverslip. 2b. Put the slide on the stage of a phase-contrast microscope set to 100, and focus on the cells. Calculate cell densities.
Each cell in a small square of a count slide is equivalent to 2 107 cells/ml. SUPPORT PROTOCOL 2

STERILITY CHECKING Although results are known only after an overnight incubation, checking the sterility of the fermentation medium prior to inoculation is essential to ensure reproducible results and aids in troubleshooting potential problems. To absolutely eliminate contamination during the test itself, it is preferable to perform this protocol under a microbiological hood. A quick check of sterility requires a sample size of 100 l, whereas more sensitive detection requires up to 100 ml. Materials Test sample (e.g., fermentation medium) LB plates with and without antibiotic (UNIT 5.2) 0.45-m membrane (Millipore, type HA or HC) Sterile filter holder For a quick check: Spread 100 l test sample onto one LB plate and one LB/antibiotic plate. Incubate at 30C overnight. Inspect for appearance of any colonies. For a more sensitive detection: Filter up to 100 ml test sample by vacuum aspiration through a 0.45-m membrane set in a sterile filter holder. Disassemble filter holder and transfer membrane to an LB agar plate, face up. Incubate plate 24 to 48 hr at 30C. Inspect membrane for appearance of any colonies. REAGENTS AND SOLUTIONS
Use Milli-Q-purified water or equivalent for the preparation of all buffers. For common stock solutions, see APPENDIX 2E; for suppliers, see SUPPLIERS APPENDIX.

Fermentation and Growth of E. coli for Optimal Protein Production

DLM medium 2.5 g adenineHCl 2.2 g L-glycine 1.3 g guanosine 1 g L-alanine 1 g L-arginine 0.8 g L-aspartic acid 0.06 g L-cystine 1.3 g L-glutamic acid 0.66 g L-glutamine 0.12 g L-histidine 0.5 g L-isoleucine 0.5 g L-leucine 0.7 g L-lysine 0.2 g L-methionine
continued

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0.26 g L-phenylalanine 0.2 g L-proline 0.2 g seleno-L-methionine 4.2 g L-serine 0.46 g L-threonine 0.34 g L-tyrosine 0.46 g L-valine 0.34 g thymine 1 g uracil 20 g glucose 0.75 g NH4Cl 1.2 g K2HPO4 0.4 g MgCl26H2O 10 ml trace element solution 1 (see recipe) H2O to 1 liter CAUTION: Seleno-L-methionine, which replaces methionine, is highly toxic. Dissolve each ingredient one after the other in water, except for L-aspartate, L-glutamate, L-tyrosine, guanosine, thymine, and uracil, which need to be dissolved first in a small amount of 0.1 N NaOH. Sterilize by filtration through a membrane with 0.22-m pores. Store at room temperature. ECPM1 medium 4 g K2HPO4 1 g KH2PO4 1 g NH4Cl 2.4 g K2SO4 132 mg CaCl22H2O 10 ml trace element solution 1 (see recipe) 20 g Casamino Acids (enzymatic hydrolysate; Difco) 3 g yeast extract (Difco) 40 g glycerol H2O to 800 ml Mix ingredients, pour into fermentor, and sterilize by heat (see Basic Protocol 1, steps 5 to 8) adding balance of water to 1 liter in step 6. Add 2 ml of 1 M MgCl26H2O (sterilized by filtration through a membrane with 0.22-m pores) as directed in step 12 of Basic Protocol 1. Once sterilized, the medium can be stored up to 1 week at 4C. HCDF1 medium 900 g glycerol H2O to 910 ml Sterilize by heat Add 20 ml 1 M MgCl26H2O (sterilized by filtration through 0.22-m membrane) Add 70 ml trace element solution 2 (see recipe)

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HCDM1 medium 10 g K2HPO4 10 g KH2PO4 9 g Na2HPO42H2O 1.1 g NH4Cl 9 g K2SO4 10 ml trace element solution 2 (see recipe) 2 g yeast extract H2O to 800 ml Sterilize by heat (see Basic Protocol 1, steps 5 to 8), adding balance of water to 1 liter in step 6. Add 2 ml 1 M MgCl26H2O (sterilized by filtration through 0.22-m membrane; see Basic Protocol 1, step 12). Supplement with carbon source (glycerol or glucose) as indicated in Alternate Protocol 1 and Alternate Protocol 3. Trace element solution 1 5 g EDTA 0.5 g FeCl36H2O 0.05 g ZnO 0.01 g CuCl22H2O 0.01 g Co(NO3)26H2O 0.01 g (NH4)6Mo7O244H2O Dissolve EDTA in 700 ml water. Dissolve each element separately by adding just enough 10 N HCl to bring it into solution. Add each element in solution one after the other to the EDTA solution. Add water to 1 liter. Adjust pH to 7.0 with concentrated NaOH. Autoclave. Store in the dark at 4C. Trace element solution 2 5 g EDTA 6 g CaCl22H2O 6 g FeSO47H2O 1.15 g MnCl24H2O 0.8 g CoCl26H2O 0.7 g ZnSO47H2O 0.3 g CuCl22H2O 0.02 g H3BO3 0.25 g (NH4)6Mo7O244H2O Dissolve EDTA in 700 ml water. Dissolve remaining ingredients separately in just enough 10 N HCl to achieve solution and add one after the other to the EDTA solution. Add water to 1 liter. Adjust to pH 7.0 with concentrated NaOH. Autoclave. Store in the dark at 4C.

COMMENTARY Background Information

The choice of a strategy for fermentation is based primarily on the type of expression system (see UNIT 5.1). For example, chemical induction (e.g., via trp, lac, or tac promoters) may be more feasible than temperature induction (e.g., via the lambda promoter) in larger-volume fermentors. The strategy also depends on the final intended use for the protein: immunization and generation of antibodies, structure-function investigations, structural studies, or in vivo assays. Because the fermentation process (i.e., the media, temperature, pH, and pO2 used) can affect the solubility and localization of the protein, it should be designed with the purification process in mind (Chapter 6). Localization of the protein in the E. coli cell (cytoplasmic, periplasmic, or secreted in the extracellular medium) is also a prime factor in

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the design of the process. Finally and most importantly, the toxicity of the target protein for E. coli is a key determinant in the success of the production. For example, cell lysis has been observed repeatedly when over-expressing proteins of the apolipoprotein family. Fermentation of recombinant E. coli has been developed by many different research groups, and several well-documented processes have been published (Knorre et al., 1991; Strandberg et al., 1991; Yang, 1992; Gram et al., 1994). In general, accurate control of environmental parameters (e.g., temperature, pH, and pO2) is prerequisite for any production. This will also ensure reproducibility among different batches. Table 5.3.1 summarizes vendors of fermentation equipment that we have used successfully. The list is not meant to be exhaustive, and local supply may vary. Although most recombinant protein production protocols described in the literature are based on a simple batch type of operation (see Basic Protocol 1), fed-batch methods (see Alternate Protocol 1) allow a more productive use of a given reactor by allowing growth of the culture to a high cell density prior to induction. The principle is to supply increasing amounts of nutrients so as to match exponential growth of the culture while minimizing accumulation of toxic by-products. Growth is made carbon sourcelimited in order to keep the concentration of residual carbon source low and to maximize yield of recombinant proteins. Fermentations may also be designed to produce labeled products. The replacement of methionine residues by selenomethionine (SeMet) in proteins (see Alternate Protocol 2) was first suggested as a new strategy to tackle a major hurdle in protein X-ray crystallography, namely, determination of the phases (Yang et al., 1990). The approach consists of using crystals of the SeMet-substituted protein in multiwavelength anomalous diffraction (MAD) experiments, which require access to a synchrotron. The SeMet protein can also be used, however, in fixed-wavelength analysis to provide landmarks (i.e., the heavy atoms of selenium) that facilitate data analysis and resolution of the structure. Nuclear magnetic resonance (NMR) spectroscopy is used increasingly for determining the three-dimensional structure of proteins (up to 40 kDa) in solution. In conventional NMR techniques, however, proteins, because of their large size, generate spectra with overlapping signals, in particular in the region of 1H resonances. This led several teams to develop a

general method for labeling proteins with different stable isotopes such as 2H, 13C, and/or 15N (see Alternate Protocol 3). Isotopic labeling allows NMR specialists to study heteronuclear coupling and to use powerful two-, three-, and four-dimensional editing of spectra. These techniques greatly facilitate assignment of resonances (for a review, see Clore and Gronenborn, 1994).

Critical Parameters and Troubleshooting

Equipment. For optimal operation, several fermentor characteristics are essential. Heat transfer is best achieved through a double jacket around a stainless steel base. Water flow should be guided in loops around the vessel wall to avoid any dead zones. Steam, if available, is the most efficient means of heating. Heating efficiency is critical for temperature shift inductions, where the broth should be heated from 30 to 42C in a few minutes. In fact, for fermentors with a working volume >10 liters, there should be provision for direct steam injection to obtain a very quick temperature shift. Cooling requires consumption of cold water, which, in some instances can be recycled. Recycling is most useful for installations where supercold water (4 to 4C) is used. In addition to the essential sensors (i.e., temperature, agitation, dissolved O2, pH, and foam level), sensors such as gas flow, pressure, load, redox potential, culture fluorescence, and mass spectrometer on exhaust gas (Todd, 1989) can be mounted to monitor the environment. Halling (1990) gives detailed information on basic principles and calibration of the essential monitors and Dusseljee and Feijen (1990) review sensors and process control information. Dissolved oxygen and O2 transfer can be controlled by agitation, air flow, gas composition, and headspace pressure. In practice, using high-agitation-rate motors with Rushton turbines and sparging O2-enriched air allow maximal transfers, but oxygen transfer can still become limiting at high cell densities. The average specific oxygen uptake rate for E. coli strains growing at a rate of 0.3 hr1 in ECPM1 medium at 30C is around 0.4 g O2/g cells/hr. Sterilization is a critical operation that can be hazardous if not properly handled. It should always be carried out according to the instructions of the fermentor supplier. An automatic controller is helpful and considerably limits the risks; it also ensures reproducibility of the operation from one batch to another. Sterility is, of course, a prerequisite for any fermentation

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Fermentation and Growth of E. coli for Optimal Protein Production

experiment. Fermentor designs should be checked carefully to ensure that all parts in contact with the culture are properly sterilized by heat. The bottom region of the reactor vessel where the agitator shaft enters the fermentor is critical. Double mechanical seals with lubrication by steam condensates provide an efficient sterility barrier. Magnetically coupled agitation systems are even safer because all seals are static. Computer control is not absolutely required for most routine purposes. It is helpful, however, for fed-batch processes and for those requiring model-based feedback control from off-line analysis. It also allows extensive data logging and analysis both from the fermentor itself and from peripheral instrumentation (e.g., off-gas analysis, flow injection analyzers). High-cell-density methods. Fed -b atch methods can be classified into two types: (i) growth-linked feeding using process parameters such as pH (Bauer and White, 1976) or pO2 (Mori et al., 1979) to control the feed of nutrients, and (ii) model-dependent feeding, controlled by a mathematical model of growth (Yee and Blanch, 1992). The first class requires little modification to a batch operation, whereas the second class necessitates on-line computer control. In the latter case, one needs to estimate growth from off-gas measurement (Reiling et al., 1985) or by real-time measurement of growth by spectrophotometry. The feed rate can be adjusted via computer control to match the increase in growth monitored by OD600. As high cell densities are obtained, limitations eventually arise from broth viscosity, oxygen transfer, or the cooling capacity of the fermentor. Figure 5.3.3 illustrates a typical fed-batch process run according to Alternate Protocol 1, with a recombinant E. coli strain and without induction of expression. Isotopic labeling. As an alternative to the defined medium described in Alternate Protocol 3, a commercial medium (Celtone) is available from Martek with labeled amino acids derived from unicellular algae. Celtone gives higher cell densities and is inexpensive and easy to make. To use this medium, follow Basic Protocol 1 for growth and induction, use 5 N NaOH for pH control when labeling with 15N, and add another 1 concentration of Celtone medium (100 ml of 10 stock solution per liter fermentation volume) at the start of induction. Harvesting. Harvesting can be achieved by tangential flow microfiltration as an alternative to continuous centrifugation (Bailey et al., 1990). Tangential flow microfiltration allows the operation to be more contained, but requires a

final centrifugation to obtain a cell cake. However, it can also be coupled to cell wash, diafiltration, buffer exchange, and cell breakage by continuous-flow high-pressure homogenizers. The most common problem found in scaling up from shaker flask experiments is the decrease in expression level. In this case, one can vary the time of induction, check plasmid stability (UNIT 5.2), and test other host/vector combinations. Changing the composition of the medium may also solve the problem but is lengthy and labor-intensive. The absence of cell lysis should be confirmed by frequent microscopic observations, and cells must be quickly harvested as soon as lysis begins. Sterility check. Because the results of this test are not known for 12 to 16 hr, the procedure is initiated and continued. In case of a contamination revealed after inoculation, the batch should be considered suspect and/or discarded. A massive contamination (which could occur if the fermentor has been prepared more than 24 hr in advance) will be obvious from the evolution of the pH and/or pO2 and turbidity of the sample. If any contamination is detected before inoculation, the entire run should be cancelled and the fermentor cleaned (see Basic Protocol 1, step 24).

Anticipated Results
With an average level of expression (e.g., 10% to 15% of total cellular proteins), a normal batch process will yield 100 g wet cell pellet per liter, containing 500 mg of the recombinant protein. This shows that even small-scale fermentations (1 to 10 liters) are quite effective in providing the starting material for purification developments. For actual production, a fed-batch process is more typically usedthis maximizes fermentor output, but also requires more manpower to operate. In this case, overall yields of 1.5 to 2 g recombinant protein per liter culture, in the crude extract, are expected.

Time Considerations
Preparation of a fermentor and its ancillaries can be done in 4 to 5 hr. Preparation of the inoculum can be started after that, allowing an overnight test for reactor sterility. The following morning inoculation can proceed, and the fermentation should be completed within 24 to 36 hr. Harvesting takes 1 to 2 hr. Analysis requires an additional 3 hr. While running the analysis, the fermentor can be cleaned, sterilized with water, and made ready for another run. Overall, a complete cycle takes 2.5 to 3 days.

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1000

OD600

100

10

100 90 80 70 60 50 40 30 20 10 0

B
Feed rate (g/liter/hr)

90 80 70 60 50 40 30 20 10 0 0
Cumulated CO2 production (mM/liter)

10

15

10,000

1000

100

10

1 0 2 4 6 8 Time (hr) 10 12 14 16

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).

Acetate (g/liter)

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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. 239:349363. 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. Schgerl, 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.

Contributed by Alain Bernard and Mark Payton Glaxo Institute for Molecular Biology Geneva, Switzerland

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