You could try adding detergents. We had a case a few years ago of an enzyme that was highly active but also highly aggregated. Addition of n-octyl beta-d-glucopyranoside significantly lowered the degree of aggregation such that crystals could be grown. Lowering the protein concentration also helped. The work is described in an Acta F paper:
Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase. White TA, Tanner JJ. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2005 Aug 1;61(Pt 8):737-9. Jack -- John J. Tanner Professor of Chemistry and Biochemistry University of Missouri-Columbia 125 Chemistry Building Columbia, MO 65211 Phone: 573-884-1280 Fax: 573-882-2754 Email: tanne...@missouri.edu http://www.chem.missouri.edu/TannerGroup/tanner.html On 8/26/09 7:55 PM, "ose toyoyuki" <o...@castor.sci.hokudai.ac.jp> 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
