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
_________________________

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