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 <http://journals.iucr.org/d/issues/2011/01/00/dz5216/index.html>Good luck. Paula On 25 January 2011 10:34, Abhilash Padavannil <g0800...@nus.edu.sg> 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 > > > > >
