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 1 M 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 1 M 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