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Preparation of competent E. coli cells Seleno-methionine labelling of proteins in E. coli


Materials

Medium A (per liter)

100 ml M9 medium (10x)
10 ml trace elements solution (100x)
20 ml 20% (w/v) Glucose
1 ml 1 M MgSO4
0.3 ml 1 M CaCl2
1 ml Biotin (1 mg/ml)
1 ml Thiamin (1 mg/ml)

appropriate antibiotic(s)


M9 medium (10x), (per liter)

80 g
 Na2HPO4
40 g
 KH2PO4
5 g
 NaCl
5 g
 NH4Cl


Trace elements solution (100x), (per liter)

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

First dissolve 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 over a 0.22 µm filter.


Stock solutions

1 M CaCl2 (sterilized)
1 M MgSO4 (sterilized)
20% (w/v) Glucose (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)
200 mM IPTG (isopropyl-b-D-thiogalactopyranoside) (filter sterilized)



Procedure

The seleno-methionine labelling of proteins is based on the use of a methionine auxotrophic E. coli strain. For vectors containing a T7 promotor we advice to use strain B834 (DE3). Initially cells are grown in minimal medium containing methionine. Before induction the cells are spun down, the pellet resuspended in mineral medium and the cells 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.

The growth rate of the culture and the optimal induction conditions can vary significantly depending on the vector, the construct and the solubility of the recombinant protein.

1. Transform the vector to the appropriate E. coli strain and plate out on minimal medium plates. Incubate the plates overnight at 37°C.
  • A transformation protocol is given in the Cloning section of the protocol database.
2. Pick one colony and use it to inoculate 5 ml Medium A plus 5 ml methionine (50 mg/ml) . Incubate the culture overnight at 37°C.
3. Add the overnight culture to 1 L medium A plus 1 ml methionine (50 mg/ml) and incubate the culture at the appropriate temperature for induction until the absorbance at 600 nm is 1.0.
4. Centrifuge the cell suspension for 10 min at 4,500 rpm (Sorvall RCB4 rotor) or 6,000 rpm (Sorvall GSA rotor) at 4°C.
5. Resuspend the pellet in 1 L Medium A (without methionine) and incubate for 4 - 8 hours at 37°C.
6. Add 1 ml seleno-methionine (50 mg/ml) and incubate for a further 30 minutes.
7. Induce overexpression of the target protein by adding 1 to 5 ml IPTG (200 mM) to the medium and incubate the culture for a further 2 to 10 hours.
8. Centrifuge the cell suspension for 10 min at 4,500 rpm (Sorvall RCB4 rotor) or 6,000 rpm (Sorvall GSA rotor) at 4°C.
9. Store the cell pellet at -20°C when not immediately used.



Working with seleno-L-methionine

Seleno-L-methionine is a very toxic compound and it should be used and disposed of carefully.

To obtain the Sigma-Aldrich Material Safety Data Sheet click here.