Hi, Chris,
My coworkers have the same problem with the ubiquitin-like protein.
Sadly, they still can't overcome this issue.
First, about autolysation: do your protein contain an any metal ions?
May be you should add EDTA to the protein buffer. Are you sure about
protein homogeneity? Is it moves as single band on SDS-PAGE?
If you generally prefer to work with 25 kDa protein:
1) try seeding!
2) what is common for all crystallization conditions? Are they PEG or
salt based? If PEG-based, then consider low salt buffer.
3) Use excessive purification: after gel filtration discard fractions
below 50-70% of the max UV absorption peak. Of course yield will drop
considerably, but it may worth it
06.05.2013 14:17, Browning Christopher ?????:
Hi Everybody,
I was wondering if anybody had a similar case to mine. The protein I'm
working on is a tough one. Its around 75 KDa in size, and it purifies
well, but soon after the full length protein starts to autolyse.
Consequently, I don't get any hits after setting up the screens.
To get around this I digest the protein with trypsin and end up with a
stable fragment of around 25KDa. When I screen this I get many hits,
but none of them look like something that can be made into single
crystals. All the hits give me large lobe-like clusters and they look
like cauliflower heads but they are optically active. I currently have
the protein in 300mM NaCl buffer. Would dropping or increasing the
salt or even using a different salt make any difference? Any ideas?
Cheers,
Chris
--
Eugene Osipov
Junior Research Scientist
Laboratory of Enzyme Engineering
A.N. Bach Institute of Biochemistry
Russian Academy of Sciences
Leninsky pr. 33, 119071 Moscow, Russia
e-mail: e.m.osi...@gmail.com
