The induction of recombinant protein expression is effected by addition of the non-metabolisable lac-inducer IPTG. Although the process is simple in theory, the BL21 cells tend to form inclusion bodies very easily. This reduces recovery dramatically and therefore in the past, each recombinant protein had to be assessed individually for their solubility and expression. This would involve optimising the length of IPTG induction and the temperature, i.e. shorter induction times and lower temperatures are considered best for maintaining recombinant solubility. However, the following two papers have shown that the existing idea that inclusion bodies are dead-end products is incorrect and, with the correct treatment, can be resolubilised, refolded and recovered as active protein. This therefore has the potential to increase recovery and do away with laborious time/temp optimisations for each recombinant protein.
- [bibliplug last_name=’Carrio’ year=’2002′]
- [bibliplug last_name=’Carrio’ year=’2001′]
Basically, the authors discovered that misfolded, insoluble proteins [inclusion bodies] formed after IPTG induction, could be resolubilised and refolded if protein synthesis was inhibited. This is achieved by adding chloramphenicol to concentrations far greater than the amount that can be degraded by the chloramphenicol acetyltransferase encoded on the pLysS plasmid. The authors also found that the chloramphenicol step should only be performed for less than 2 hours [for the protein they were studying] as longer incubation times resulted in protein loss.
- 5 ml LB in universals, containing kanamycin or ampicillin PLUS chloramphenicol.
- 1 L LB in a 5 l Erlenmeyer flask [no antibiotics added]
- Stock kanamycin, ampicillin and chloramphenicol.
- 500 mM IPTG stock solution in distilled water [you will need 2 ml of this for a 1 l culture]. You can make this stock solution in advance and store at 20°C until required.
- 500 ml LB [no antibiotics added].
- Philips spectrophotometer
- Plastic, disposable cuvettes
- Sterile pipettes for sampling culture for OD readings.
- You can either streak out a scraping from a glycerol stock onto LB-agar + antibiotics (kanamycin and chloramphenicol, both are poured on top of the agar which is then allowed to dry for 5 minutes before streaking), or you can scrape with a sterile tip and drop into a 5 ml LB solution. Incubate at 30°C overnight. If using a 5 ml culture, ensure that the bottle is shaken gently and is inclined, but not completely horizontal.
- If you grow colonies overnight on LB-agar, then select a single colony and inoculate into the 5 ml culture. Grow up overnight at 30°C. At the same time, make 1 L LB broth and place it in the 37°C incubator overnight to equilibrate.
- The overnight culture should be turbid (appear cloudy but not too cloudy) after overnight incubation.
- Add kanamycin or ampicillin to the 1 L LB broth to the appropriate concentration but don’t add any chloramphenicol. If you add chloramphenicol in the bulking up step, your resultant cell density will be reduced. There is no reason to have chloramphenicol in the broth at this stage.
- Dilute 1 ml overnight culture into the 1 L LB media [1:1000 dilution]. Grow with vigorous shaking at 37°C. Leave for at least 6 hours. When you start seeing turbidity, remove an aliquot and check the OD at 600 nm, using LB [no cells but with antibiotics] as a blank. The literature recommends inducing at an OD of 0.4-0.6, but 0.8-1.0 OD gives a higher yield.
- Once the correct OD has been reached, remove the culture from the 37°C incubator and transfer to the 30°C incubator. Add IPTG to [final] = 1mM. Allow to shake vigorously overnight [~ 12 h].
- Pellet the cells by centrifugation at 4 000 x g, 20 min, RT. Resuspend in 500 ml LB. Add chloramphenicol to a final concentration of 200 μg/ml [from a 34 mg/ml stock solution in 100% ethanol].
- Shake at 37°C for 2 hours.
- Pellet the cells by centrifugation at 4000 x g, 20 min, 4°C. Resuspend the pellet in buffer [the type of buffer depends on the protein; in most cases, the same buffer that is used for introducing the protein to the ion exchange column, but without salt, is used] and prepare for lysis.