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Hi, This isn't the first and I suspect not the last case like that - sometimes 'soluble' proteins require detergents to be 'happy' upon overexpression. We've had several such cases - to me the most memorable one being that of the catalytic domain of PTPbeta, expressed in E. coli as a His-MBP fusion. Upon cleavage (TEV or TVMV protease), the protein requires 0.01% bOG to be present in solution, otherwise it irreversibly precipitates. PTPbeta-CD crystallizes perfectly fine, and the crystals diffract to high resolution - and there are no signs of detergent in any of the structures solved so far, nor do we feel that the fold was affected in any way (based on comparisons with other phosphatase structures etc.). Unless you feel that your detergent concentration is excessive, I don't perceive this as a reason to be concerned. If you're concerned about detergent concentration, you can always pick one that can be dialyzed or one that forms micelles at high concentration - obviously there's no guarrantee that the substitute detergent works out. Alternatively you can attempt to substitute NDSBs for detergents, engineer your protein to be more soluble, etc. etc. Artem _____ From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Daniel Jin Sent: Tuesday, August 28, 2007 1:45 PM To: [log in to unmask] Subject: [ccp4bb] water soluble protein that needs detergent to be stable Hi, I am working on a 60 kDa C. elegans protein that is predicted to be mostly alpha-helix. It is over-expressed in E.coli and the yield is about 1 mg/L of cell culture. The CD spec at 4 degree showed the presence of dominant alpha-helix. However, we don't have any functional assay to confirm that it is folded correctly. It is over-expressed as a GST-fusion. We noticed that after cleavage of GST, it will easily precipitate if moved to room temperature (solution turns cloudy). Otherwise, it is OK at 4 degree. The CD temperature melting experiment showed a gradual change of signal, no sharp transition was observed. We later found out that including some detergent in the buffer will make it stay soluble at room temperature and showed as a dimer on SEC (4C or RT). Glycerol at 10% will help too but not as good as detergent. My concerns are, first this protein might not folded correctly, second, the presence of probably high concentration of detergent in the final sample will harm crystallization since the detergent will be co-concentrated with the protein. I am wondering whether anyone has deal with proteins like this before and their experience on improvement of the biochemical behavior and of course crystallization. Many thanks. Best, chen _____ Got a little couch potato? Check out fun summer <http://us.rd.yahoo.com/evt=48248/*http:/search.yahoo.com/search?fr=oni_on_m ail&p=summer+activities+for+kids&cs=bz> activities for kids.