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Expression of SeMet labeled proteins: Difference between revisions

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Several methods are available for the bacterial expression of semet-labelled proteins.
= bacterial expression =
 
 
 
references:


In the literature:


Original literature source for met synthesis silencing (for use with semet labelling of non-auxotrophs):
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).
Van Duyne et al. J. Mol. Biol. 229, 105–124 (1993).


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


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


Online:
Online:


Two protocols for semet labelling as of 11/03/2008:
Two protocols for semet labelling as of 11/03/2008:
http://www.doe-mbi.ucla.edu/local/protocols
http://www.doe-mbi.ucla.edu/local/protocols


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:
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:
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400 ml M63 stock salts
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]
# 20 ml of solution 1


20 ml of [http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_SeMet_labeled_proteins#solution_2: solution 2]
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


20 ml of [http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_SeMet_labeled_proteins#solution_3: solution 3]
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 [http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_SeMet_labeled_proteins#solution_4: solution 4]
# 20 ml of solution 3


20 ml of [http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_SeMet_labeled_proteins#solution_5: solution 5]
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
 
# 20 ml of solution 4
 
for 50 ml
200 mg hypoxanthine
200 mg uracil
dissolve in 50 ml DMSO
 
# 20 ml of solution 5
 
for 500 ml (in ddH<sub>2</sub>O)
100 mg biotin
100 mg nicotinamide
10 mg riboflavin
100 mg thiamine
 
# M63 stock solution
 
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


2 ml of 1 M MgSO<sub>4</sub> stock
2 ml of 1 M MgSO<sub>4</sub> stock
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== M63 stock solution: ==
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 1: ==
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
== solution 2: ==
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
== 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 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.
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