1. Using the same E. coli host strain as that used in the previous culture grown in regular growth medium. Also use the same plasmid construct. The expression host does not have to be methionine auxotroph.
2. Grow the overnight culture and the final fermentation culture in M9 minimal medium1 containing glucose (0.4% w/v) and the appropriate amount of antibiotic(s).
3. Grow the fermentation culture until the OD600 reaches 0.8 (OD600 at 0.6 if in shake flask culture).
4. At this point, add the SeMet amino acid cocktail2.
5. 15 minutes later, add IPTG to Cf=1mM for induction.
6. Grow the culture for whatever period of time that gives the best expression.
Solution Preparation
1. M9 minimal medium
When cooled, per liter of culture volume, add the following pre-0.2um-filter-sterilized solutions:
0.4% glucose (4g/L dextrose to give Vf = 0.4% ) or glycerol (50mL/L to give Vf = 5% glycerol) as the carbon source
2. SeMet amino acid cocktail
60mg Selenomethione, along with
100mg lysine hydrochloride
50mg leucine
Add about 10mL of water and vortex (about 5-10 min) to dissolve.
Per liter of culture volume, add:
6.8g anhydrous form Na2HPO4, or 12.8g Na2HPO4. 7H2O
3g KH2PO4
0.5g NaCl
1g NH4Cl
QS with distilled water to 90% of the final volume and then autoclave at 15 lb/sq for 15 min.
Note: glycerol can actually be autoclaved
2mL of 1M MgSO4
0.1mL of 1M CaCl2
100ul of 0.5% thiamine (Vitamin B1)
QS to Vf = 1 liter
Per liter of culture volume, add:
100mg threonine
100mg phenylalanine
50mg isoleucine
50mg valine
Sterilize by 0.2um-filtration when actually add to culture.
Reference: Van Duyne, Gregory D., Standaert, Robert F., Karplus, P. Andrew, Schreber, Stuart L., Clardy, Jon (1993), Atomic Structures of the Human Immunophilin FKBP-12 Complexes with FK506 and Rapamucin. J. Mol. Biol. 229. 105-124.