Expressing proteins in E. coli

Janet Smith | Boston Biomedical Research Institute

These protocols have been used successful for proteins expressed in a variety of pET expression vectors from Novagen.

Culture Growth and Induction

  1. Prepare 1 L of LB in a 4 or 6L flask, and 25 ml LB in a 250 ml flask.
  2. Inoculate the 25 ml LB + selective antibiotic(s) flask with a single freshly-grown colony containing the plasmid of interest. Fresh transformants are sometimes better, but this is usually not necessary. Shake @ 37°C overnight with cap loosened to increase aeration.
  3. The next morning add the selective antibiotic(s) to the 1L LB culture, and inoculate with the entire 25 ml overnight culture by simply pouring it in. Shake @ 200 RPM and 28.5°C (for soluble proteins) or 37°C (for insoluble proteins.) Many proteins that are soluble at lower temperatures become insoluble above 30°C, so it is important to carefully monitor the temperature.
  4. Check OD600 every hour or so. A disposible plastic cuvette and water as a blank are fine for this.
  5. When the OD600 is between 0.3 and 0.6, remove a 5 ml sample and place in a sterile test tube. This is the uninduced control; continue to incubate it with the big culture. Induce the large culture by adding IPTG to 0.4 mM (MW of IPTG = 238.3 g, add 0.0953 g to a 1L culture.)
  6. Allow to grow for 3-5 hrs before harvesting.

Harvesting

  1. Prior to harvesting, place a 1.5 ml aliquot of the induced cells in an eppendorf tube. This will be used for a gel sample to check the induction. Store on ice, along with the uninduced control.
  2. Harvest cells by centrifuging @ 7,000 RPM, 4°C for 7 min in four 250 ml centrifuge bottles.
  3. Pour off the supe and invert the bottles on a paper towel. After a few minutes, remove excess supe from the inside of the bottle by wiping it with a paper towel, staying far away from the pellet.
  4. Resuspended in the cells in Binding Buffer (20 mM Tris-HCl, pH 7.9, 5 mM imidazole, 500 mM NaCl), using a volume equal to 1/50th the volume of the original culture. (For a 1 L culture, resuspend the pellet in each of the four centrifuge bottles in 5 ml.)
  5. Transfer the resuspended pellets to 15 ml conical tubes and store @ -80°C.

Induction Check

This is a quick way to check how well the protein expressed, but it is necessary to follow up with a check of the solubility of the protein using the next protocol, as well.

  1. Take 200 μl aliquots from the uninduced and induced samples.
  2. Centrifuge the aliquots in a microfuge at top speed for 2 min. Remove the supe by aspiration, being careful not to disturb the pellets.
  3. Resuspend the pellets in 100 μl of 1X SDS sample buffer, boil for 5 min, and then place back on ice.
  4. The samples can be stored @ -20°C. Load 15 μl of each sample on an SDS PAGE gel. The induced protein is usually readily apparent as a band of the appropriate size in the induced culture.

Solubility Check

  1. Spin down 1 ml of the induced and uniduced cultures for 2 min at top speed in a microfuge.
  2. Aspirate off supe; respin for 5 s and remove remaining supe with a micropipet.
  3. Resuspend cell pellets with 200 μl Binding Buffer, and freeze the samples at -80°C.
  4. Thaw the samples. They should be quite viscous, indicating that the cells have lysed.
  5. Add

    2 μl 1M MgCl2
    4 μl DNAse I solution*
    2 μl 100 mM PMSF

    and incubate at RT until the samples become less viscous. This can be assayed by pipeting up and down with a P200.
    * DNAse I solution = 1 mg/ml DNAse I (bovine pancrease Grade II, Boehringer Mannheim) in 50% glycerol, 20 mM Tris-HCl, pH 7.5, 1 mM MgCl2; store at -20°C
  6. Add 3 μl lysate to 47 μl 1X SB. These are the "whole cell" samples.
  7. Spin 5 min at top speed in a microfuge at 4°C. Add 3 μl supe to 47 μl 1X SB. These are the "soluble" samples.
  8. Remove supe, wash pellets by vortexing with 1 ml cold lysis buffer, respin, and remove supe.Dissolve pellets by boiling in 200 μl 2% SDS. It may be necessary to vortex and pipet up and down a bit to get it to dissolve.
  9. Add 3 μl dissolved pellet to 47 μl 1X SB. These are the "insoluble" samples.
  10. Boil samples 5 min. Load 10 μl on a 10-well minigel.