Expression of SeMet labeled proteins: Difference between revisions
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= bacterial expression = | |||
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: | |||
for | Inoculate 2 l of the following media with 8 ml of seed | ||
culture (grown for 4 hours at 37°C in 2xYT): | |||
1475 ml ddH<sub>2</sub>O | |||
400 ml M63 stock salts | |||
# 20 ml of 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 | |||
# 20 ml of 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 | |||
# 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 | |||
# 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 | 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 | |||
20 ml of 20% glucose stock | |||
8 ml of ampicillin stock (100 mg/ml) | |||
100 mg | |||
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 in other systems = | |||
* 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. | |||
* Yeast | |||
* 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. |
Latest revision as of 08:29, 24 March 2010
bacterial expression[edit | edit source]
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
- 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
- 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
- 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
- 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 ddH2O) 100 mg biotin 100 mg nicotinamide 10 mg riboflavin 100 mg thiamine
- 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 in other systems[edit | edit source]
- 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.
- Yeast
- 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.