5
edits
Changes
From CCP4 wiki
Jump to navigationJump to searchExpression of SeMet labeled proteins (edit)
Revision as of 01:10, 4 November 2008
, 01:10, 4 November 2008no edit summary
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
Inoculate 2 l of the following media with 8 ml of seed
culture (grown for 4 hours at 37°C in 2xYT):
culture (grown for 4 hours at 37°C in 2xYT):
