Protein expression

Estimated time

2 days

Protocol

Note

The BL21 strain of E. coli is best for protein production. It has several proteases knocked out, among other modifications. There are several sub-strains that can be relevant:

  • BL21: The classic, useful if your promoter is a standard E. coli promoter such as lac, tac, trc, ParaBAD, PrhaBAD, or T5.

  • BL21(DE3) : A strain with integrated T7 polymerase. Can be used in place of BL21 but is required if using the T7 or T7-lac promoters.

  • BL21-RIL : A strain with extra copies of certain tRNAs, in order to correct for the rarity of some codons in E. coli. This can be helpful in upping protein titers, especially when expressing some mammalian proteins.

  • Rosetta2 (DE3) : A strain with integrated T7 polymerase, plus extra tRNAs and other modifications that make it ideal for protein expression.

  1. In the morning, start a 5 mL culture of your strain, in LB/antibiotic media.

  2. Prepare TB media in Erlenmeyer flasks. For proper aeration, flasks should be filled to at most 30% the listed volume.

  3. Before leaving for the day, incoluate 20 mL LB/antibiotic cultures with 400 uL of the 5 mL culture.

  4. The following morning, use the NanoDrop to calculate the OD of the overnight cultures. Inoculate large flasks to an OD600 of 0.075 (e.g. three doublings to get to 0.6).

Note

Remember to take a sample of LB/TB media to properly blank the NanoDrop!

Induction to OD600 0.075 means that each 20 mL culture can induce around 1L of TB media, as the overnight OD600 should be around 5-10. If you are inducing more than 1L, you may need to make multiple 20 mL cultures.

Calculate OD’s with the simple dilution formula:

\[\text{mL starter culture} = \frac{\text{mL bulk culture} \cdot 0.075}{\text{OD of starter culture}}\]

  1. Grow cultures until an OD600 of 0.6. Sample every half hour to an hour. It should take around 90 minutes to 2 hours to reach and OD of 0.6.

  2. Lower the temperature to 30C and induce with 0.5 mM sterile-filtered IPTG.

  3. Let the culture grow for 5-6 hours.

  4. Harvest cells via centrifugation at 4000xg for 30 minutes. At this point, the cell pellet is immediately ready for further protein purification steps. The cell pellet can also be stored at -20C or -80C.