Protein production¶

Overview¶

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A large amount of protein can be produced by introducing a protein encoding plasmid into a BL21(DE3) E. coli* strain containing a DE3 element that expresses T7 RNA polymerase.

In this setup, the gene must be under a T7 promoter in the lac operon. Protein production is induced with IPTG that binds LacI (lac repressor) and thus opens up RNA polymerase's access to the promoter. IPTG is not consumed and thus protein production remains stable. In practice, a lacUV5 promoter is used, which is stronger that the wild-type lac promoter. IPTG needs to be optimized in order to balance the amount of protein produced in such a way that it does not overwhelm a cell.

Protein production can further be stabilized by introducing a pLysS plasmid that constitutively expresses low levels of a T7 lysozyme. T7 lysozyme inhibits basal levels T7 RNA polymerase, thus protein expression is suppressed even if the T7 promoter is leaky. This is especially useful the the protein is toxic to the host cell.

Protein production is typically done in two phases: 1. Biomass growth at optimal conditions (37C) 2. Protein production at less favorable conditions (e.g., 16C)

When the amount of the resulting protein is unknown a priori, it is recommended to grow about 3 L of medium to obtain a large amount of biomass in a single run and have enough material even for small amounts of protein.

Reagents and materials¶

Materials¶

Note that the protocol is set up for 8 flasks. This may seem as an overkill but it saves you from running the procedure again in case your protein of interest is poorly expressed.

Day	Material	Vol.	Quantity	Purpose
1	Dual-antibiotic plate (CHL + specific antibiotic for your plasmid)		1	Plating
2	Conical flask	2 L	8	TB
	Conical flask	500 mL	1	TB
	Small bottle	500 mL	1	TB for dilutions
	Bottle	1 L	1	10X phosphates
	Serological pipette	25 mL	1	10X phosphates
3	Conical falcon tube	500 mL	4	Pelleting
	Serological pipette	50 mL	1	10X phosphates
	Serological pipette	5 mL	1	Culture
	Cuvette	1 mm	5	OD measurements
	Eppendorf microtube	1.5 mL	2	Culture samples for SDS PAGE

Reagents¶

CHL – 8 x 400 uL -> 3.5 mL
CB – 8 x 400 uL -> 3.5 mL
1 M IPGT – 8 x 40 uL -> 350 uL

TB media¶

Component	Amount to get	Final conc. (g/L)
Yeast extract	74.4 g	24
Tryptone	37.2 g	12
Glycerol	15.62 g	5.04
dH2O	up to 3.1 L

Combine in a large plastic container with a handle – it will make it easier to pour.
Initially dilute in a smaller volume (e.g., 80%) and stir by hand until the powders have fully solved. It will look like beer with a bit of foam.
Add the remaining water.
Autoclave at 1/20' (121C).

10X phosphates¶

Component	Amount to get	MW (g/mol)	Final conc. (M)
KH2PO4	9.25 g	136.09	.17
K2HPO4	50.16 g	174.18	.72
dH2O	up to 400 mL

Filter sterilize or autoclave at 1/20' (121 C).
Mixing phosphates with TB before autoclaving may yield precipitation. Typically still works but most conservative practice is to add sterilized/autoclaved phosphates to the TB on the day of usage.

Procedure¶

Day 1: Transformation (optional)¶

In the evening, transform the plasmid containing protein-expressing gene into Bl21(DE3) pLysS and plate on a dual-antibiotic plate* with CHL (for pLysS) and the specific antibiotic for your plasmid.

Day 2: Overnight growth¶

(Optional) In the morning, pick a colony from the transformation plate and spread it on a fresh dual-antibiotic plate (CHL + specific antibiotic).
Prepare TB medium components:
1. Autoclave TB medium (without phosphates):
  1. 360 mL per 2 L flask (8 flasks total),
  2. 90 mL for the overnight culture in a 500 mL flask.
  3. The remaining TB volume (~100 mL) in a small bottle (for OD600 measurements).
2. Prepare 10X phosphates solution (KH2PO4 and K2HPO4). Autoclave them separately (or add them to the TB medium via a filter on the day of use).
In the evening, to the 90 mL TB medium flask add:
1. 10 ml of 10X phosphates;
2. 100 uL of CHL (for pLysS);
3. 100 uL of the specific antibiotic for your plasmid.
Set up overnight culture in the TB flask.

Day 3: Biomass growth (3-4 hours)¶

Make sure the overnight culture has grown (should look cloudy).
Outgrowth:
1. To each 2 L flask with TB medium add:
  1. 40 mL 10X phosphates;
  2. 400 uL of CHL (for pLysS);
  3. 400 uL of the specific antibiotic for your plasmid.
2. Transfer 1 mL from one of the medium flasks to a cuvette for blanking. Cover with foil.
3. Add 4 mL overnight culture to each 2 L flask.
4. Measure the initial OD600 by sampling from one 2 L flask.
5. Grow in a shaker (250 rpm, 37C) until reaching OD600 .5-.6.
  - Measure every hour and estimate when the required OD600 will be reached by computing the doubling time.
  - Start measuring more often closer to the target OD600 so that you don't overgrow. Cells grow fast in the log phase!
6. Cool down to room temperature or lower before induction.
Protein production:
1. Put aside 1 mL of the culture before induction for SDS-PAGE.
2. Add 400 uL of 1 M IPTG (final concentration – 1 mM) and grow shaking at 250 rpm. Choose the temperature and duration according to the properties of your protein, but a safe choice is 18C for 24 h.
  - Final IPTG concentration typically is between 100 uM and 1 mM and depends on your protein / plasmid.

Day 4: Pelleting¶

Cool down the culture flasks at 4C for at least 10 min.
Cool down centrifuge to 4C and set 500 mL conical tubes under UV.
Take 1 mL of the resulting culture, dilute it 10X with 9 mL of TB medium, and measure OD600.
Take another 1 mL of the resulting culture and dilute it to the same OD600 you had just prior the IPTG induction. Put aside 1 mL for SDS-PAGE.
Pelleting:
1. Transfer into 4 x 500 mL conical tubes from 4 flasks, spin down for 12 min 4000 rpm at 4C, and discard supernatant.
  - Make sure you counterbalance them accurately by equalizing their weight.
  - Place the remaining flasks in 4C.
2. Transfer the remaining flasks contents to the conical tubes (pouring on top of the pellet), spin down for 12 min 4000 rpm at 4C, and discard supernatant.
3. Store the conocals upright at -20C overnight.
  - Alternatively, for more sensitive proteins, consider snap-freezing in liquid nitrogen and storing in -70C. However, in this case you'll need to scrape the pellet into a 50 mL falcon, which is a messy process and you lose some pellet, and then taking the pellet out when you start purification becomes more cumbersome as well.
4. Weigh a piece of aluminum foil, then place the conicals upside down under running room temperature water for the pellet to fall out, and place each pellet on the foil. Store at -20C.
Preparation for SDS-PAGE:
1. Split each 1 mL sample that you put aside into 100 uL and 900 uL.
2. Spin down the samples at 3500 g for 2 min and discard supernatant.
3. Resuspend with a small amount of Milli-Q and measure the total volume by aspiring with pipette.
4. Add the correct proportion of 4X SDS loading dye and heat at 98C for 5 min.
  - The samples should be fairly homogeneous. If you get a viscous clump instead, you may have sampled too much culture and got too much genomic DNA. Do not use such concentrated samples for SDS-PAGE as they will not run properly.
5. Freeze to -20C or run SDS-PAGE immediately.