We would like to thank you for all the responses so far, and to post a
follow up.
Some of you have suggested that lysis is not the problem, but rather the
presence of inclusion bodies is keeping the solution from changing
appearance. We do run a sample of the pellet on SDS-PAGE after every
purification and we never detect significant amounts of our protein in it,
so we had initially ruled out the possibility of inclusion bodies coming
into play.
However, we will certainly look at the pre- and post-lysis solutions under
the microscope to look for evidence of lysis, as was suggested initially.
Depending on the outcome we will consider using a BeadBeater or Lysozyme
treatment, as was also suggested.
In response to your question, our cell cracker is an AH-2010 Nano
Homogenize Machine from ATS Engineering.
We appreciate your suggestions and will post a summary (complete with
acknowledgements) as soon as we reach a conclusion.
Thanking you,
--
J. Valencia S.
PhD student
CGR-NU
> 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
>
>
