Hi Toyoyuki, If your protein bind to metal ions you could try low concentration of chelating agents in the purification and storage buffer. Take a look at the following reference:
Chelating Agents Stabilize the Monomeric State of the Zinc Binding Human Papillomavirus 16 E6 Oncoprotein Degenkolbe et al., Biochemistry, 2003, 42 (13), pp 3868–3873 Few years back, based on the above mentioned paper, keeping low amount of EGTA (note: NOT eDta) and DTT helped me to stabilize a Zinc binding RING domain as dimer instead of soluble aggregate. Of course, every protein is different but above reference might help you. If you lucky enough to identify a chelating agent that prevent aggregation of your protein, you might also try to include that chemical in the expression media. Good luck and all the best, -Partha Partha Sampathkumar NYSGXRC, Lilly Biotechnology Center San Diego CA 92121 PS: At that time I purified this particular RING domain over NiNTA column. Since both EGTA and DTT are not good for the NiNTA resin I kept required higher concentration of both waiting for the protein in the 1.7ml eppendroff tubes that were used to collection elution fractions. On Sat, Aug 29, 2009 at 7:52 PM, Xuan Yang <pattisy...@gmail.com> wrote: > Dear James, > > Could you provide the reference of your success story? My protein also > formed large soluble aggregates and I am desperate for such successful > stories! > > By the way, have your performed DLS to your protein? What about the > polydispensity? Is it lower than 20%? > > Thanks in advance! > > Sincerely, > > Xuan Yang > 2009/8/27 James Stroud <xtald...@gmail.com> > >> Just try crystallizing it. What is a crystal but a "massive aggregate"? >> That they are still soluble and active is great news. >> >> As a grad student, I had a similar phenomenon with an early project. I >> showed a gel in group meeting where both activity and aggregation were >> obvious, said the aggregate was no problem, got ridiculed when I said I was >> going to throw it in trays despite what anyone said, had giant crystals >> after a few trays, and solved the structure with miras. >> >> Get the structure and then worry about why it's aggregating. The structure >> will probably provide you with the clues you need. >> >> >> On Aug 26, 2009, at 5:55 PM, ose toyoyuki wrote: >> >> >>> Dear all, >>> >>> This is a question on how to cope with the protein that seems to form >>> massive aggregates in solution but enzymatically active. >>> >>> I'm working on a protein whose molecular weight is around 70kDa and can >>> be >>> divided into two domains (say A and B domains). We expressed this protein >>> in >>> E.coli fused with GST and purified using some chromatography. The GST >>> affinity chromatography works well and proteinase digestion to remove the >>> tag does wonders too. The purified protein was confirmed to be active >>> enough, we can detect both activities from these two domains. But the >>> retention time from the gel filtration clearly shows it is awfully >>> aggregated (comes out at the void region). DLS measurement indicates the >>> averaged diameter is around 45 nm, which I feel is a bit too long. >>> Analytical ultracentrifuge result implies that the distribution of the >>> molecular species is wide, some portion got precipitated with 1K rcf >>> (means >>> the molecular weight is more than 5MDa) and the rest is ranging from 1MD >>> to >>> 5MDa with a peak at 1MDa. >>> I made new two constructs covering the A and B domains respectively, both >>> of >>> them are active again, but only the A domain has got the same symptom as >>> the >>> intact protein. The B domain seems to exist as a monomer in solution. >>> >>> Here come my questions, (I) How can I interpret this phenomenon? (II) Is >>> there anything we can try to change the situation? (III) Does it make >>> sense >>> to try crystallization? (probably not).(IV) Has anyone got such >>> experience? >>> >>> I tried the methylation on lysine side chains, I also tried the buffer >>> with >>> 0.2M arginine or 10% glycerol but the all the results just seem hopeless. >>> The protein before the proteinase treatment also comes out at the void >>> region from the gel filtration. >>> >>> >>> cheers >>> >>> toyoyuki >>> >>> --? >>> Toyoyuki Ose >>> o...@sci.hokudai.ac.jp >>> >>> Graduate School of Life Science >>> Hokkaido University >>> N21W11 Kita-ku, Sapporo >>> 001-0021 Japan >>> >> >
