Difference between revisions of "Expression of SeMet labeled proteins"

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Several methods are available for the bacterial expression of semet-labelled proteins.
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= bacterial expression of semet-labelled proteins =
  
  
In the literature:
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 +
references:
  
  
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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]
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# 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]
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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]
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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]
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# 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]
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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
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# 20 ml of solution 4
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for 50 ml
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200 mg hypoxanthine
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200 mg uracil
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dissolve in 50 ml DMSO
 +
 
 +
# 20 ml of solution 5
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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)
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15 g KH<sub>2</sub>PO<sub>4</sub>
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45.6 g K<sub>2</sub>HPO<sub>4</sub>
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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
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= expression of semet-labelled proteins in other systems =
100 mg nicotinamide
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* 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
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* Yeast
100 mg thiamine
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* stable S2 insect cells
 +
* baculovirus expression system

Revision as of 07:23, 24 March 2010

bacterial expression of semet-labelled proteins

references:


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).


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


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


Online:


Two protocols for semet labelling as of 11/03/2008: 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:

___________________________________________________________


Protocol:

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

  1. 20 ml of 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

  1. 20 ml of 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
  1. 20 ml of 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

  1. 20 ml of solution 4
for 50 ml
200 mg hypoxanthine
200 mg uracil
dissolve in 50 ml DMSO
  1. 20 ml of solution 5
for 500 ml (in ddH2O) 
100 mg biotin
100 mg nicotinamide
10 mg riboflavin
100 mg thiamine
  1. 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

2 ml of 1 M 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



expression of semet-labelled proteins in other systems