I have not dealt myself with this type of protein, but Alex McPherson tested the detergent beta-octylglucoside (at a concentration of 1.5 %, i.e. above the cmc) for the crystallisation of soluble protein and tRNA (1986, J.Biol.Chem 261:1969-75), the detergent did not hampered the crystallisation, and even sometimes improved the crystal quality. It is possible to somewhat control the detergent concentration: if the cmc is high enough (something above 1 mM) you can dialyse the detergent, otherwise you can bind the protein to a small affinity column and djust the required concentration of detergent. I stop here because I find always very difficult to extrapolate the effect of detergent from membrane protein to soluble protein. Daniel
Daniel Jin a écrit : > 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 activities for kids. > <http://us.rd.yahoo.com/evt=48248/*http://search.yahoo.com/search?fr=oni_on_mail&p=summer+activities+for+kids&cs=bz> >
