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.
