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:
-ml ddH<sub>2</sub>O
-ml M63 stock salts
-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).
-ml of solution 2
-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
-ml of solution 4
-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
-ml of 1M MgSO<sub>4</sub> stock
-ml of 20% glucose stock
+Online:
-ml of ampicillin stock (100 mg/ml)
-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
-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):
-g KH<sub>2</sub>PO<sub>4</sub>
+ml ddH<sub>2</sub>O
+ml M63 stock salts
-.6 g K<sub>2</sub>HPO<sub>4</sub>
+# 20 ml of solution 1
-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
-.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
+mg phenylalanine
+mg tryptophane
+mg tyrosine
+mg p-aminobenzoic acid
+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
+mg hypoxanthine
+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)
+mg biotin
+mg nicotinamide
+mg riboflavin
+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)
+g KH<sub>2</sub>PO<sub>4</sub>
+.6 g K<sub>2</sub>HPO<sub>4</sub>
+g (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
+.5 ml FeSO<sub>4</sub> at 2.5 mg/ml
-== solution 3: ==
+ml of 1 M MgSO<sub>4</sub> stock
-for 50 ml
+ml of 20% glucose stock
-mg phenylalanine
+ml of ampicillin stock (100 mg/ml)
-mg tryptophane
-mg tyrosine
-mg p-aminobenzoic acid
-mg p-hydrobenzoic acid
-dissolve in 50 ml 0.1 M KOH
+ml of L-SeMet stock (10 mg/ml)
-== solution 4: ==
+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
-mg hypoxanthine
+induce protein expression
-mg uracil
+harvest cells 20 hours later
-dissolve in 50 ml DMSO
-== solution 5:  ==
-for 500 ml (in ddH<sub>2</sub>O)
-mg biotin
+= expression in other systems =
-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.
-mg riboflavin
+* Yeast
-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.