Table of Contents
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Overexpression of folded soluble protein in E. coli liquid cultures
Pilot scale overexpression
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 ug/mL ampicillin into a sterile 10 mL (17 x 100 mm) culture tube.
- Inoculate the medium with 100 uL of a fresh, overnight E. coli culture harboring the appropriate overexpression plasmid.
- Incubate with shaking (250 rpm) for 2½-3 hr. Take a 1 mL sample of cells, spin down gently and store at -20C for later PAGE analysis.
- 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. Remove 1 ml aloquots at appropriate 1-2 hour intervals, spin down and store cell pellets frozen.
- Perform SDS-PAGE analysis of whole cells: resuspend the cell pellets collected in 30uL lysis buffer for 20 minutes. Spin down cells for 10 minutes at >11,000 rpm and collect supernatant. Combine with loading dye and run PAGE gels as described.
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 LB medium containing 100 ug/mL ampicillin into a sterile 300 mL culture flask. (see below)
- 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% NaCl (see below)
- Take up wet cell pellet (typically 0.5-3 g) in 15 mL of the appropriate extraction buffer(see below) and freeze-thaw to lyse cells.
- Analyze expression via SDS-PAGE analysis.
Notes
- 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.
- Inclusion of 3 mM arabinose sugar in autolytic cells removes the need for a beat beater or sonication step: 90% of cells will lyse after a single freeze-thaw cycle.
- The saline wash is optional, but may reduce protein contamination from the growth medium.
- A suggested buffer is 20 mM TrisCl-100mM NaCl-10 uM EDTA, pH 8.0. Typically, protease inhibitors such as PMSF are added to limit proteolysis of the desired protein, and 5-10% glycerol is often desirable in an initial buffer. The exact composition of the buffer will depend on the optimum conditions required for the protein of interest.
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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 (we typically use XJb cells or other BL21 derivative) 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 ug/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 sterile TB medium containing the appropriate antibiotic to 2.5-3.0 L culture flasks.(see below)
- 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.(see below)
- Grow cells 6 hr to overnight with vigorous shaking at 25-37 °C.(see below)
- 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
- 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.
- Inclusion of 3 mM arabinose sugar in autolytic XJb cells removes the need for a beat beater or sonication step: 90% of cells will lyse after a single freeze-thaw cycle.
- 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 uM of metal ion should be sufficient for even the most robust metalloenzyme expression system.
- 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.
- The appropriate post-induction growth temperature should be determined by experiment. Inclusion body formation is often reduced by overexpression at lower temperatures.
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Optimized Overexpression for DesC
The following protocol has been shown to optimize the over-expression of DesC in the soluble fraction of the cell culture. Reference the DesC project notebook to see diagnostic protocol and SDSPage gel.
- Innoculate a 10mL volume of LB medium with a single colony of BL21 transformants.
- Grow overnight the 10mL volume of cells in a 15mL culture tube at 37°C
- Warm 1L of TB medium at 37°C and centrifuge the 10mL of cell culture at 2500rpm for 10minutes, discard the supernatant and resuspend cells in 2-4mL of TB medium.
- Add 100ug/mL ampicillin and 3mM arabinose to the TB medium and innoculate with the resuspended cells.
- Place Tunair flasks with cell culture in 37ºC at 215rpm until cell culture, when measured at 600nm in the UV spec, is between 0.5 and 0.6 absorbance units.
- Innoculate culture with 0.5mM IPTG
- Place cell culture back at 215rpm in 15ºC for 4-6 hours.
Notes
If induced cell culture is allowed to incubate overnight the majority of the desired over-expressed protein will be trapped in inclusion bodies (the insoluable fraction) and a successful purification will not be possible, limiting the post-induction incubation to 4-6 hours alleviates this complication
Optimized Overexpression for DesD
The following protocol has been shown to optimize the over-expression of DesD in the soluble fraction of the cell culture. Reference the DesD project notebook to see diagnostic protocol and SDSPage gel.
Prep for overexpression:
Make 1n L of TB broth recipes and autoclave, prepare DesD plasmid molecular biology from DH5a cells (~1/semester) and transform XJb/BL21 cells molecular biology.
The day before overexpression:
- Innoculate a 10mL volume of LB w/ amp medium with a single colony of BL21 transformants.
- Incubate and shake O/N in a 15mL culture tube at 37°C
The day of overexpression:
- Warm 1L of TB medium at 37°C and centrifuge the 10mL of cell culture at 2500rpm for 10minutes, discard the supernatant and resuspend cells in 2mL of LB medium.
- Add ampicillin and arabinose to final concentrations of 100ug/mL and 3mM respectively to each liter of TB media recipes, remove 1 mL of TB medium to use as a blank for cell growth measurements, and then innoculate the 1 L sample of TB medium with the resuspended cells.
- Place Tunair flasks with cell culture in 37ºC at 215rpm until cell culture, when measured at 600nm in the UV spec, is between 0.5 and 0.6 absorbance units (usually around 3-5 hours, often closer to 3)
- Innoculate culture with 0.5 mM IPTG
- Place cell culture back at 215rpm in 15ºC for overnight growth.
Notes for E.coli Expression
- To "grow overnight" generally means to grow for 8-16 hours.
- When planning cell growths, try to plan the timing so that cultures do not exceed the 16 hour growth time, as this can lead to development of natural Ampicillin resistance.
- Grow an 8-10 mL overnight culture for every cell pellet you wish to make.
- Cover ABS blank cuvette with a parafilm to avoid contamination.
Cell-Free (AKA "cellfie") Overexpression
The following protocol is adapted from GenScript's protocol GenScript Cell-Free Kit Instruction Manual