Expression of SeMet labeled proteins: Difference between revisions

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Expression of SeMet labeled proteins (edit)

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Inoculate 2 l of the following media with 8 ml of seed
= bacterial expression =
culture (grown for 4 hours at 37°C in 2xYT):
 
 
 
references:


1475 ml ddH<sub>2</sub>O
400 ml M63 stock salts


20 ml of [http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_SeMet_labeled_proteins#solution_1: solution 1]
Original literature source for met synthesis silencing (for use with semet labelling of non-auxotrophs):
Van Duyne et al. J. Mol. Biol. 229, 105–124 (1993).


20 ml of solution 2


20 ml of solution 3
Protocols for both auxotrophs and non-auxotrophs, as well as some other non-bacterial systems:
Doublié, S. (1997). Methods Enzymol. 276, 523-532


20 ml of solution 4


20 ml of solution 5
Autoinduction semet protocol (allegedly complicated recipe):
Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207-234


2 ml of 1M MgSO<sub>4</sub> stock


20 ml of 20% glucose stock
Online:


8 ml of ampicillin stock (100 mg/ml)


10 ml of L-SeMet stock (10 mg/ml)
Two protocols for semet labelling as of 11/03/2008:
http://www.doe-mbi.ucla.edu/local/protocols


8 ml seed culture


aliquote the 2 liter solution into 8X 250 ml
Another non-auxotroph protocol is copied below, courtesy of G. Fritz. Fritz says this recipe could probably be made in one vessel for immediate use, except for the aromatic aa's which might dissolve better at basic pH:
grow at 37°C over night until OD600=0.400


induce protein expression
___________________________________________________________
harvest cells 20 hours later




== M63 stock solution: ==
Protocol:


for 1 liter (in ddH2O)
Inoculate 2 l of the following media with 8 ml of seed
culture (grown for 4 hours at 37°C in 2xYT):


15 g KH<sub>2</sub>PO<sub>4</sub>
1475 ml ddH<sub>2</sub>O
400 ml M63 stock salts


45.6 g K<sub>2</sub>HPO<sub>4</sub>
# 20 ml of solution 1


10 g (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
for 50 ml (in ddH<sub>2</sub>O)  
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
# 20 ml of solution 2


2.5 ml FeSO<sub>4</sub> at 2.5 mg/ml
for 50 ml (in ddH<sub>2</sub>O)
add 200 mg of the following amino acids:
N, D, C, E
pH should be adjusted to 7-7.5 with 1 M KOH


# 20 ml of solution 3


== solution 1: ==
for 50 ml
200 mg phenylalanine
100 mg tryptophane
200 mg tyrosine
20 mg p-aminobenzoic acid
20 mg p-hydrobenzoic acid


for 50 ml (in ddH<sub>2</sub>O)
dissolve in 50 ml 0.1 M KOH


add 200 mg of the following amino acids:
# 20 ml of solution 4
A, R, G, Q, H, I, L, K, P, S, T, V


pH should be adjusted to 7-7.5 with 1 M KOH
for 50 ml
200 mg hypoxanthine
200 mg uracil
dissolve in 50 ml DMSO


== solution 2: ==
# 20 ml of solution 5


for 50 ml (in ddH<sub>2</sub>O)
for 500 ml (in ddH<sub>2</sub>O)  
100 mg biotin
100 mg nicotinamide
10 mg riboflavin
100 mg thiamine


add 200 mg of the following amino acids:
# M63 stock solution
N, D, C, E


pH should be adjusted to 7-7.5 with 1 M KOH
for 1 liter (in ddH<sub>2</sub>O)
15 g KH<sub>2</sub>PO<sub>4</sub>
45.6 g K<sub>2</sub>HPO<sub>4</sub>
10 g (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
2.5 ml FeSO<sub>4</sub> at 2.5 mg/ml


== solution 3: ==
2 ml of 1 M MgSO<sub>4</sub> stock


for 50 ml
20 ml of 20% glucose stock


200 mg phenylalanine
8 ml of ampicillin stock (100 mg/ml)
100 mg tryptophane
200 mg tyrosine
20 mg p-aminobenzoic acid
20 mg p-hydrobenzoic acid


dissolve in 50 ml 0.1 M KOH
10 ml of L-SeMet stock (10 mg/ml)


== solution 4: ==
8 ml seed culture


for 50 ml
aliquote the 2 liter solution into 8X 250 ml
grow at 37°C over night until OD600=0.400


200 mg hypoxanthine
induce protein expression
200 mg uracil
harvest cells 20 hours later


dissolve in 50 ml DMSO


== solution 5:  ==


for 500 ml (in ddH<sub>2</sub>O)


100 mg biotin
= expression in other systems =
100 mg nicotinamide
* mammalian cells: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2242577/pdf/2008.pdf mentions several mammalian cell lines, including CHO, HEK239, and COS. Apparently, Lec8 have been used, too.
10 mg riboflavin
* Yeast
100 mg thiamine
* stable S2 insect cells
* baculovirus expression system
* CHO LecR cells, early reference (1994) from Wayne Hendrickson's lab (PMID: 7922031)
* probably the easiest labelling method for HEK293 cells can be found in PMID: 17001101.
Kay
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