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.  
 
Several methods are available for the bacterial expression of semet-labelled proteins.  
 +
  
 
In the literature:
 
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:

Revision as of 01:11, 4 November 2008

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


In the literature:


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

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 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


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