Methods S3: Conditions for seleno-methionyl protein preparation in fed batch
cultivation
One liter of synthetic medium contained YNB 13.4 g, glycerol 40 g, a phosphate
buffer pH 6 0.1M, trace elements 1X PTM1 [H3BO3 0.02 mg, CuSO4.5H2O 6 mg, KI 0.1 mg,
MnSO4.H2O 3 mg, Na2MoO4 0.2 mg, ZnCl2 20 mg, FeSO4.7H2O 65 mg and H2SO4 (96%)
0.002 mL] and biotin 0.4 mg. The temperature was controlled at 28°C. A pulse with an
identical concentrated medium was given upon depletion of glycerol. Induction by methanol
was then initiated for 4 days with a constant (2 mL/h) or gradual supply (2 to 7 mL/h) of the
synthetic feed-medium. The temperature was decreased to 22°C. Selenomethionine (to a final
concentration of 250 mg/L) and amino acids (400 mg/L of partial inhibitors of the methionine
pathway: leucine, isoleucine, valine tryptophan, phenylalanine, and growth activators: lysine,
alanine, arginine, aspartic acid, glycine, asparagine, proline, serine, threonine, histidine,
glutamine, glutamic acid) were added in one or four pulses according to the experiments.