Hi,

I often see no real change in change in solution appearance after sonication mediated lysis, with proteins which yield low amounts or no soluble protein in E. coli. I've had a look at the solution post lysis under the microscope and the cells are infact lysed, it's just the presence of high levels of inclusion bodies means the solution remains turbid. Check your pre and post lysis solution under the microscope to see if you see the same thing.


Cheers,

Rhys

________________________________________
From: CCP4 bulletin board [[log in to unmask]] On Behalf Of J. Valencia S. [[log in to unmask]]
Sent: 21 June 2012 13:44
To: [log in to unmask]
Subject: [ccp4bb] Expressed protein hinders cell lysis?

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