Dear Linda As we described, we used an adapted minimal media to introduce SeCys into our recombinantly expressed protein. As a SeCys source, we used seleno-cistine (provided by Sigma) which is reduced to seleno-cysteine and incorporated into the proteins. It works really well and incorporation levels were high, without the need for any significant changes to the rich media protocol to produce protein. If you have a protein rich in Cys which you can currently express in E. Coli, it is worth trying this simple approach. You can then try SAD or MAD.
As for the more complicated methods, we didn't try them, so can't comment. Good luck! If you have any questions, don't hesitate to contact me, I'd be happy to help. HNY! Best regards Paula =================================== Dr Paula S. Salgado Lecturer in Macromolecular Crystallography Institute for Cell and Molecular Biosciences Faculty of Medical Sciences 3rd Floor Cookson Building Newcastle University Newcastle upon Tyne, NE2 4HH, UK Tel: +44 (0)191 222 5810 Fax: +44 (0)191 222 7424 Email: paula.salg...@ncl.ac.uk ________________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Linda Olson [lol...@mcw.edu] Sent: 26 December 2012 18:33 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] SeCys usage for SAD Dear All, I have read a recent paper by Salgado et al about using non-auxotrophic E.coli to incorporate SeCys into recombinant protein for phasing purposes. Does anyone have a source for Selenocysteine? I have also seen a paper by Schaefer et al which uses nitric acid treated elemental Se for a sulfur surrogate to generate Se-labeled protein. Has anyone else tried this? My proteins are rich in Cys and tend to lack Met so the prospect of labeling cys is very attractive. Thanks, Linda ________________________ Linda Olson, PhD Research Scientist II Dept Biochemistry Medical College of Wisconsin 8701 Watertown Plank Rd Milwaukee, WI 53226 phone: 414-955-8545 fax: 414-456-6510 _________________________
