General Considerations

It is highly recommended that protein overexpression be carried out in protease deficient overexpression strains like BL21 (suitable for pTrc99 or plasmids with E. coli lac or trc promoters) or BL21(DE3) (suitable for pET vectors or other T7 lac promoter plasmids). Having stated that, we have performed protein expression in cloning strains like JM109 without observing proteolysis. If not using protease deficient strains for overexpression, the use of low temperature, protease inhibitors, and immediate purification after cell breakage is highly recommended.

Pilot scale overexpression

It is frequently advisable to assess protein overexpression levels obtained with a plasmid/host combination before committing to large scale overexpression trials. The following protocol is convenient for preliminary assessment of overall protein overexpression in E. coli harboring vectors with trc or lac promoters and an ampicillin resistance gene.

• Using sterile technique, transfer 2 mL of LB medium containing 100 μg/mL ampicillin into a sterile 10 mL (17 x 100 mm) culture tube.
• Inoculate the medium with 100 μL of a fresh, overnight E. coli culture harboring the appropriate overexpression plasmid.
• Incubate with shaking (250 rpm) for 2½-3 hr.
• Using sterile technique, add IPTG to a final concentration of 0.2-1.0 mM, to induce overexpression.
• Incubate with shaking (250 rpm) for 6 hr to overnight.
• Perform SDS-PAGE analysis of whole cells as described previously.

In order to determine what, if any, fraction of the overexpressed protein is produced in soluble form, it is necessary to grow a slightly larger culture in order to produce cell-free extracts. This is conveniently done immediately following the preliminary pilot overexpression.

• Using sterile technique, place 100 mL of TB medium containing 100 μg/mL ampicillin into a sterile 300 mL culture flask.¹
• Using sterile technique, pipet 1.0 mL of fresh, overnight E. coli culture into each of two (2) sterile 1.5 mL microcentrifuge tubes.
• Centrifuge at 6000 xg for 3 minutes, remove the supernatant, and resuspend in 1.0 mL of sterile growth medium.
• Centrifuge at 6000 xg for 3 minutes, remove the supernatant, and wash once again with 1.0 mL of sterile growth medium.
• Resuspend cells in each tube with 1.0 mL of sterile growth medium, and use the contents of both tubes (2.0 mL) to inoculate the 100 mL of growth medium previously prepared.
• Incubate with shaking (250 rpm) until A₆₀₀ reaches 0.6-1.0, about 2½-3 hr.
• Using sterile technique, add IPTG to a final concentration of 0.2-1.0 mM, to induce overexpression.
• Incubate with shaking (250 rpm) for 6 hr to overnight.
• Harvest cells by centrifugation at 8000 xg for 5 min in two 50 mL centrifuge tubes.
• Wash cells twice by resuspension and centrifugation in cold 0.9% NaCl²
• Take up wet cell pellet (typically 0.5-3 g) in 15 mL of the appropriate extraction buffer³ and place in the small Bead Beater chamber.
• Break open cells in the Bead Beater and perform SDS-PAGE analysis

Notes

1. We highly recommend Tunair culture flasks (IBI Scientific) for large-scale cell culture. These flasks have excellent baffling and aeration and typically produce 5x the cell mass of typical culture flasks.
2. The saline wash is optional, but may reduce protein contamination from the growth medium.
3. A suggested buffer is 20 mM MOPS-1 mM DTT-10 μM EDTA, pH 7.0. Typically, protease inhibitors are added to limit proteolysis of the desired protein. The exact composition of the buffer will depend on the optimum conditions required for the protein of interest.

Production-scale overexpression

The large-scale overexpression of target proteins typically requires 1-4 L of cell culture. Such cultures cannot be started directly from single colonies on agar plates, but must be scaled up gradually. The following protocol is typical for most overexpression systems, and should observe proper sterile technique:

• The desired strain of E. coli harboring the desired overexpression plasmid should be streaked out on an agar plate containing the appropriate antibiotic and grown overnight.
• A single colony should be used to inoculate 10 mL of LB medium containing 100 μg/mL ampicillin or appropriate antibiotic, and the culture shaken overnight at 37 °C. Alternatively, cells can be grown until A₆₀₀ reaches 0.6-1.0, and then stored at 4 ºC for up to 24 hr.
• For each 1 L of overexpression culture, place 10 mL of overnight scale-up culture in a 50 mL centrifuge tube.
• Pellet cells at 8000 xg for 5 min, and wash with 10 mL fresh LB medium with antibiotic as described above.
• Resuspend washed and pelleted cells in 10 mL of fresh LB medium.
• Prepare overexpression cultures by adding 1 L of TB medium containing the appropriate antibiotic to 2.5-3.0 L culture flasks.¹
• Inoculate each 1 L of overexpression medium with the entire contents (10 mL) of one 50 mL centrifuge tube.
• Incubate at 37 °C with vigorous shaking for 2½-3 hr, or until A₆₀₀ reaches 0.6-1.0, then induce by adding IPTG to a final concentration of 0.2-1.0 mM.^2,3
• Grow cells 6 hr to overnight with vigorous shaking at 25-37 °C.⁴
• Harvest cells immediately by centrifugation at 8000 xg in 500 mL centrifuge bottles, or store at 4 °C for no more than a few hours before processing. The typical yield of wet pelleted cells is 20-30 g per liter of culture for cultures grown at 37 ºC.
• If pelleted cells are not to be processed immediately, store at -80 °C.

Notes

1. We highly recommend Tunair culture flasks (IBI Scientific) for large-scale cell culture. These flasks have excellent baffling and aeration and typically produce 5x the cell mass of typical culture flasks.
2. If metal ions are required for overexpression, e.g. for the overexpression of metalloenzymes, add them at the time of induction. Many metal ions will inhibit cell growth, resulting in lengthy pre-induction growth phases. A final concentration 10-100 μM of metal ion should be sufficient for even the most robust metalloenzyme expression system.
3. IPTG is hideously expensive. We have found that most expression systems using the trc or T7 promoters express protein more than adequately at 0.2 mM IPTG.
4. The appropriate post-induction growth temperature should be determined by experiment. Inclusion body formation is often reduced by overexpression at lower temperatures.

Operating the Genesys 10uv Spectrophotometer

In order to measure absorbance at 600nm, the following protocol is appropriate.

• Turn the machine on approximately 10 minutes prior to use. It will take some time for it to calibrate.
• Select utility -> stored test directory -> A600 -> run test.¹
• At this point, the instrument will bring up two options: run blank and run sample. Open the machine and insert your blank into the box marked "B" and your sample to be measured into the box marked "1."
• Run the blank first (making sure that the cuvette inserted is facing the light source) followed by the sample.
• A general rule of thumb is that the sample's absorbance will double every half hour (if your sample's initial absorbance is 0.050 at t=90 minutes, then at t=120 minutes, the sample's absorbance will be 0.100 and so on).
• Once your absorbance exceeds 0.600, it is appropriate to inoculate your culture with the appropriate amount of IPTG.

Notes

1. For every instance of ->, press enter.

Harvesting overexpression cultures

The extraction of overexpressed soluble proteins from E. coli cultures requires removal of the culture medium, breakage of cells, and clarification of the extract. All operations should be carried out at 4 °C or with samples placed on ice to minimize protein denaturation and proteoloysis. For cell breakage we are currently using a BeadBeater™ (Biospec) which efficiently disrupts cells by agitation with small glass beads. A typical protocol follows:

• In tared 500 mL centrifuge bottles, pellet cells by centrifugation at 8000 xg for10 min, discarding the supernatant
• Weigh the bottles to determine the mass of wet, pelleted cells.
• Resuspend cells in no more than 40 mL¹ of extraction buffer by scraping, vortexing, and/or shaking vigorously.
• Combine resuspended cells in one container, and add any protease inhibitors desired.
• Fill the pre-chilled homogenzation chamber of the BeadBeater™ half-full with 0.1 mm glass beads, and add resuspended cells.
• Top off the chamber with additional extraction buffer so that there will be no air space left when the cap is attached.²
• Disrupt cells 15 seconds, followed by a 45 second rest period to re-chill the homogenization chamber contents. Repeat this disruption/rest cycle a total of 8 times for complete homogenization.³
• Load the crude cell extract into the required number of appropriately balanced 50 mL centrifuge tubes.
• Centrifuge at 48000 xg for 40-60 min to clarify the cell extract.
• Carefully decant the supernatant for purification.⁴

Notes

1. The medium chamber of the Bead Beater can accommodate about 40-45 mL of cell suspension when properly filled with glass beads. If using the small chamber (suitable for 100 mL expression culture, use no more than 15 mL of extraction buffer.
2. It is critical that there is no air in the homogenization chamber. Air in the chamber will reduce disruption efficiency and may denature extracted protein.
3. If using the large (350 mL) homogenization chamber, use cycles of 1 minute disruption and 3 minutes rest. You shouldn't have to use this chamber unless you are harvesting cells from 8-10 L of culture!
4. It may be necessary to filter the supernatant through a 0.45 μm syringe filter prior to FPLC purification to prevent the tiny glass beads from damaging the instrument.

Thrombin cleavage of purification tags

Many proteins are conveniently expressed with purification tags, e.g., 6x His-tags, maltose binding protein, glutathione-S-transferase, etc. to simplify purification to homogeneity and/or to improve expression levels of soluble protein. These tags must often be removed before the protein of interest can be studied. One of the most common removal methods involves proteolytic cleavage of the purification tag utilizing a cloned thrombin recognition site. A typical protocol for thrombin cleavage of a tagged protein using a Novex Thrombin Cleavage Capture Kit follows:

• In a 2.0 mL microcentrifuge tube add
  • ≈2 mg tagged protein (up to 1600 μL)
  • 200 μL 10x thrombin cleavage buffer
  • water to a total volume of 1800 μL
• Add 200 μL diluted biotinylated thrombin¹ and mix gently by inversion
• Incubate at room temperature for 2-24 hours²
• Remove 2-5 μL for later SDS-PAGE analysis
• Thoroughly resuspend streptavidin agarose slurry by inversion and pipet 32 μL into reaction mixture with a wide-bore pipet tip³
• Incubate for 30 min at room temperature. Mix gently by inversion every few minutes to keep streptavidin agarose resuspended
• Transfer no more than 350 μL at a time of the reaction mixture to the sample cup of a spin filter
• Centrifuge at 500 xg for 30 s. Remove filtrate with a transfer pipet to a clean 2.0 mL microcentrifuge tube
• Repeat the previous two steps until all of the reaction mixture is processed, taking care to save the filtrate solution, which is your cleaved protein
• Store protein at 4 ºC and purify protein as soon as practical using gel exclusion chromatography

Notes

1. The exact dilution of thrombin should be determined by prior cleavage trials, and could vary from 1:20 to 1:400 dilution. To minimize non-specific cleavage, the greatest dilution of thrombin should be used that gives complete cleavage in the desired time interval.
2. The exact incubation time should be determined by prior cleavage trials. Utilize the shortest cleavage time that results in complete cleavage using the desired thrombin dilution.
3. In the absence of wide-bore pipet tips, cut off a portion of the end of a standard pipet tip to accommodate passage of streptavidin agarose particles