While staying on the subject, I found the following paper very useful
when trying to produce Se-Met labeled protein in HEK293 Flp-In.
Barton et al. Highly efficient selenomethionine labeling of
recombinant proteins produced in mammalian cells. Protein Sci (2006)
vol. 15 (8) pp. 2008-13
Vangelis
--
Vangelis Christodoulou
Analist C
Anastassis (Tassos) Perrakis lab
The Netherlands Cancer Institute (NKI) B8.038
Plesmanlaan 121, 1066 CX, Amsterdam
The Netherlands
tel: +31 (0)20 512 1963
fax: +31 (0)20 512 1954
Website: xtal.nki.nl/Tassos_group/
On 4 Nov 2008, at 03:27, Artem Evdokimov wrote:
http://www.xtals.org/pdfs/selenium.pdf
(sorry, for some reason first email went out w/o the hyperlink)
Artem
-----Original Message-----
From: Artem Evdokimov [mailto:[EMAIL PROTECTED]
Sent: Monday, November 03, 2008 9:26 PM
To: 'Jacob Keller'; 'CCP4BB@JISCMAIL.AC.UK'
Subject: RE: [ccp4bb] Semet in non-auxotrophic strains (summary)
By popular request I've placed our modified Se-Met method online:
-----Original Message-----
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf
Of Jacob
Keller
Sent: Monday, November 03, 2008 8:13 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Semet in non-auxotrophic strains (summary)
Hello all,
Prompted by Kay Diederichs, herein follows a summary of the responses
gleaned from the CCP4BB. The wiki has also been edited to include the
summary:
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_Se
Met_labeled_proteins
Summary:
1. Protocols for both auxotrophs and non-auxotrophs:
Doublié, S. (1997). Methods Enzymol. 276, 523-532
2. Two protocols for semet labelling:
http://www.doe-mbi.ucla.edu/local/protocols
3. Another non-auxotroph protocol. G. 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:
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_Se
Met_labeled_proteins
4. Original literature source for met synthesis silencing (for use
with
non-auxotrophs):
Van Duyne et al. J. Mol. Biol. 229, 105-124 (1993).
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
Jacob Keller
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED]
*******************************************
