Expression of SeMet labeled proteins
Inoculate 2 l of the following media with 8 ml of seed culture (grown for 4 hours at 37°C in 2xYT):
1475 ml ddH2O 400 ml M63 stock salts 20 ml of solution 1 20 ml of solution 2 20 ml of solution 3 20 ml of solution 4 20 ml of solution 5 2 ml of 1M MgSO4 stock 20 ml of 20% glucose stock 8 ml of ampicillin stock (100 mg/ml) 10 ml of L-SeMet stock (10 mg/ml) 8 ml seed culture
aliquote the 2 liter solution into 8X 250 ml grow at 37°C over night until OD600=0.400 induce protein expression harvest cells 20 hours later
M63 stock solution:
for 1 liter (in ddH2O)
15 g KH2PO4 45.6 g K2HPO4 10 g (NH4)2SO4 2.5 ml FeSO4 at 2.5 mg/ml
solution 1:
for 50 ml (in ddH2O)
add 200 mg of the following amino acids: A, R, G, Q, H, I, L, K, P, S, T, V
pH should be adjusted to 7-7.5 with 1M KOH
solution 2:
for 50 ml (in ddH2O)
add 200 mg of the following amino acids: N, D, C, E
pH should be adjusted to 7-7.5 with 1M KOH
solution 3:
for 50 ml
200 mg phenylalanine 100 mg tryptophane 200 mg tyrosine 20 mg p-aminobenzoic acid 20 mg p-hydrobenzoic acid
dissolve in 50 ml 0.1 M KOH
solution 4:
for 50 ml
200 mg hypoxanthine 200 mg uracil
dissolve in 50 ml DMSO
solution 5:
for 500 ml (in ddH2O)
100 mg biotin 100 mg nicotinamide 10 mg riboflavin 100 mg thiamine