I'm not convinced you are not getting lysis but if you want to try something different use a BeadBeater. They are gentle and very efficient.
Roger Rowlett On Jun 21, 2012 8:54 AM, "J. Valencia S." <[email protected]> wrote: > Greetings, everyone. We need to ask your advice on an issue with one of > our proteins expressed in E. coli Rosetta cells. This yeast-derived > protein has a very low yield compared to others we work with, and we think > it is because the cells are hard to lyse: even after 3 cycles in a cell > cracker the solution barely changes colour. > > We have no problems lysing Rosetta cells expressing other yeast-derived > soluble proteins, and we usually obtain enough for our crystallisation > screens. For the aforementioned protein we have already tried using STAR > cells, varying the contents of the lysis buffer, sonicating, or adding > FeSO4 to the solution (we think the protein binds Fe or Mn because it is > yellow), but to no avail. > > Searching the ccp4bb archive and other resources did not help, so we would > like to ask 2 questions to the community in order to focus our efforts > better: > 1. How can a recombinant protein make a cell harder to lyse? > 2. Do you have any suggestions to avoid this effect? > > We appreciate any input, and will be sure to post a summary for future > reference once this issue is solved. > > Sincerely, > > > -- > J. Valencia S. > PhD student > CGR-NU >
