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
