Seleno-methionine labelling

This method is shamelessly filched from van den Ent et al., (1999) Structure Fold Des. 7(10):1181-7.
It works by feedback inhibition of the methionine biosynthesis pathway and does not require a methionine auxotrophic strain.
This variation relies on auto-induction and does not require IPTG.

For 20°C induction

Pick a single, freshly-transformed BL21 DE3(RIL) colony into 1 litre M9 media plus . . .
- 0.4 % glucose (determine optimal concentration empirically)
- 2 mM MgSO₄
- 100 µg ml^-1 ampicillin (as appropriate)
Grow overnight at 37°C until thick
Cool to 20°C and take sample if required
Sub-culture at 1:50 (i.e. 20 ml in 1 litre) into M9 plus goodies (as above) and 1/100 trace elements and grow for 4 - 5 hours at 20°C
To each litre add
- 100 mg L-selenomethionine
- 100 mg L-lysine
- 100 mg L-threonine
- 100 mg L-phenylalanine
- 50 mg L-leucine
- 50 mg L-isoleucine
- 50 mg L-valine
Grow overnight at 20°C until thick and expressing target protein.

Protein preparation

Harvest and resuspend cells in the normal way, if possible working slighter faster than normal
After lysis and during preparation maintain 5 mM DTT in all buffers
Store in 5 - 10 mM DTT. Grow crystals as soon as possible.

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