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Why do you need a fusion protein? Can you use His-tag directly? If you have to have a fusion protein, use one that has no hydrophobic patches on the surface, because otherwise they will be glued to each other. For separation of your mixture that you have now try dilute it as much as possible ( but keep it detectable) and hopefully the complex might dissociate. I also think that adding some organic solvent might help to dissociate it. Then you apply to your column. Maia ----- Original Message ----- From: Celina R. To: [log in to unmask] Sent: Monday, May 24, 2010 6:05 AM Subject: [ccp4bb] question - GFP fusion - cleavage sites 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.