Hi, I have been trying to express a GST-fusion protein in E. Coli and after GST-sepharose column, I've found that on a SDS-page gel, in addition to the band corresponding to my GST-fusion protein, there is an addition band pretty close to and underneath it, presumably this represents the partial degraded product of the above protein (and the second band persists after protease digestion to remove the GST part). Since GST itself exists as dimer and in fact I know the protein fused to GST also exists as dimer, is it possible to add a reagent, if there is any, into the buffer that disrupts dimer formation of any sort, so that I can then run some kind of chromatography to separate the "full length" protein from the partial degraded product? And what kind of chromatography should I used. Kindly appreciate your advice in advance

I'm no wizard with the chromatography of proteins, but I've done some work in the past with reasonable success. I would expect that one would need to know what kind of bonding is causing the dimerization. If it's a disulfide linkage, wouldn't dithiothreitol or beta-mercaptoethanol disrupt these bonds? It's been a while since I've worked with proteins, so please forgive if I'm stating the obvious here.

In any case, depending upon the size of the protein, you could try HPLC equipped with a reversed phase column (C4, C8, or C18)with a 300 angstrom pore and a gradient between 94.9:5:0.1 Water:MeCN:TFA (Trifluoroacetic acid) and 99.9:0.1 MeCN:TFA to resolve the peptides. Detection would be done via UV-vis.

I hope this is helpful. If you'd like to talk further, please e-mail me at christopher.judd@collabo.com

Best,

Chris

If the dimerization is not due to disulfide formation you might succeed in dissociating the dimer with a chaotropic medium in either RP or high resolution exclusion chromatography. The subject is complex, so check books, the web, etc., on chaotropic/lyotropic series and phenomena. Just one note of caution: salts which are listed as lyotropic might be quite chaotropic at lower concentration. Feel free to e-mail me, I have learned some regarding protein chrom. but also need to learn more myself.
Another point: We have had tremendous problems with TFA and other perfluoro acids, and will use them only as a last resort.
Hans