Hi Celina, I cannot answer to your question concerning the GFP-related problem. Fot the thrombin vs TEV protease related question I can tell you that in my hands thrombin works really very well in most detergents (TEV is somehow more sensitive). I am working on membrane proteins purified in very different detergents such as OG, DM, DDM, FosCholines and mixed lipids/detergents mixture and I see good (very good) cleavage with thrombin. We buy it from Sigma ( bovine thrombin) and it can be stored for years at -20C in a suitable buffer without loss of efficiency.
Hope this helps, Cheers Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu n Mon, May 24, 2010 at 5:05 AM, Celina R. <r.g.cel...@gmail.com> wrote: > Dear CCP4er's, > Sorry for the non-crystallography related question and was hoping someone > on the bulletin board might have some suggestions to overcome my peculiar > protein purification problem. > > I am working on several membrane proteins (for crystallization trials) that > have a C-Terminal eGFP fusion partner followed by a His-tag. The membrane > protein and the GFP-His tag are separated by a TEV protease site. After > purifying the fusion protein by IMAC, I add TEV protease to cleave the > linkage between the membrane protein and the GFP-His tag.The cleavage > reaction is also dialyzed to get rid of the imidazole. This cleavage seems > to go to completion as judged by SDS-PAGE. However, when I try to separate > the membrane protein from the GFP-His tag by passing through a IMAC column > twice (excess nickel resin), a significant amount (about 1 mg) of the > GFP-His tag doesn't bind the IMAC column and flows through along with my > protein. In addition, other methods such as centricons (30, 50 or 100 kDa > M.W.C.O.), Gel Filtration and Ion-Exchange are also not able to separate > them. All my buffers have 5 mM reducing agent and 500 mM NaCl to try and > prevent any non-specific interaction between my protein and the GFP-His tag. > It appears that the GFP-His tag is somehow stuck to my protein and co-elutes > on any chromatographic column that i use. > > Has anyone encountered such a problem and managed to overcome it? Any > suggestions/tricks would be helpful. > I also have a question: Which is better to use for the cleavage of > His-tags, in case I want to clone the membrane protein without GFP: TEV > protease or thrombin? > > Thanks in advance. > C. > --
