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
