Before starting:

• Prepare all required reagents and solutions

Protocol:

The seleno-methionine labelling of proteins is based on the use of a methionine auxotrophic E. colistrain. For vectors containing a T7 promotor we advice to use the E. coli strain B834(DE3). Initially cells are grown in minimal medium containing methionine. Before induction the cells are spun down, the pellet is resuspended in minimum medium and the cells are starved before the addition of seleno-L-methionine. Then overexpression of the target protein is induced and it will incorporate seleno-L-methionine. In some cases the addition of all essential amino acids during the initial growth phase (step 2 & 3) improves results.

To test if the strain is really auxotrophic pick one colony from a minimal medium plate containing methionine to inoculate 5 ml Medium A without methionine. Incubate the culture overnight at 37°C. With an methionine auxotrophic strain no growth should be observed.

1. Transform the vector into an appropriate E. coli expression strain and plate out on minimal medium plates; incubate overnight at 37°C
2. Pick a colony and use this to inoculate 5 ml of Medium A plus 5 µl Methionine (50 mg/ml); grow overnight at 37°C
3. Add the overnight culture to 1 liter of Medium A plus 1 ml Methionine (50 mg/ml) and grow the culture at the appropriate temperature until the OD₆₀₀ ~ 1.0
4. Centrifuge the cell culture for 10 min. at 4000 rpm and 4°C
5. Resuspend the cell pellet in 1 liter Medium A (without Methionine) and grow for 4-8 hours at 37°C
6. Add 1 ml Seleno-L-Methionine (50 mg/ml) and grow for an additional 30 min. at 37°C
7. Induce expression of the target protein and continue culturing for 2-12 hours
8. Harvest the cells by centrifugation at 4°C
9. When you don’t continue immediately with the protein purification, freeze the cell pellet and store at -20°C until further usage

Comment: the growth rate of the culture and the optimal induction conditions can vary significantly depending on the vector backbone, the construct design and the solubility of the protein of interest

Medium A (per liter):

• 100 ml M9 medium (10x)
• 10 ml trace elements solution (100x)
• 20 ml 20% (w/v) glucose
• 1 ml 1M MgSO₄
• 0.3 ml 1M CaCl₂
• 1 ml biotin (1 mg/ml)
• 1 ml thiamin (1 mg/ml)
• Appropriate antibiotics

10x M9 medium (per liter)

• 60 g Na₂HPO₄
• 30 g KH₂PO₄
• 5 g NaCl
• 5 g NH₄Cl

100 x trace elements solution (per liter):

• 5 g EDTA
• 0.83 g FeCl₃.6H₂O
• 84 mg ZnCl₂
• 13 mg CuCl₂.2H₂O
• 10 mg CoCl₂.6H₂O
• 10 mg H₃BO₃
• 1.6 mg MnCl₂.6H₂O

Comment: First dissolve the 5 g EDTA in 800 ml water and adjust the pH to 7.5. Then add the other components and adjust the volume to 1 L. Sterilize the solution by filtration through a 0.22 µm filter.

Stock solutions

• 20% (w/v) glucose (sterilized)
• 1M MgSO₄ (sterilized)
• 1M CaCl₂ (sterilized)
• 1 mg/ml Biotin (filter sterilized)
• 1 mg/ml Thiamin (filter sterilized)
• 50 mg/ml Methionine (filter sterilized)
• 50 mg/ml Seleno-L-Methionine (filter sterilized)