I think the likely reason here are the points where you did the
mutations having adverse effects
- how do the mutants behave without labeling? this would be of course
an important control...
-Tommi
On 25.1.2011, at 12.52, Paula Salgado wrote:
Dear Abhilash
You could try an alternative labelling option: if your protein has
cysteines, try labelling those with selenium. We succeeded in doing
this using non-auxotrophic strains, meaning you deviate less from
your native expression protocol and are therefore more likely to
obtain well folded and soluble sample, in the correct dimeric form.
For details of our protocol, see:
http://journals.iucr.org/d/issues/2011/01/00/dz5216/index.html
Good luck.
Paula
On 25 January 2011 10:34, Abhilash Padavannil <[email protected]>
wrote:
Dear All,
I am working on structure determination of a bacterial protein. The
protein was expressed as a GST fusion protein in E. coli. I got
crystals of the native protein.
I am trying to label my protein with Se-Met.
The purification protocol involves GST affinity purification,
cleavage of the fusion protein followed by gel-filtration. The
native protein behaves well and on gel filtration majority of the
protein elutes as a dimer with very little eluting in the void volume.
However, using the same protocol with the Se-Met protein, most of
the protein elutes in the void volume with very little or nothing as
a dimer. I am struggling with this for quite some time. I also tried
by adding 10mm DTT in the buffers only to see that the fusion
protein does not bind to the beads at this concentration of DTT.
Could someone suggest me on how to increase the proportion of
dimeric protein?
I have one more question, does the position of methionines in the
protein effect its hydrophobicity? For the sake of labeling I have
mutated a few leucines in the native protein to Met and they all lie
very close to one another. Could this be the reason for heavy
aggregation?
Thanks in advance,
Abhilash
