Hi, 182kDa is large I'd say, but not too large for crystallisation.
Since you are using a Ni-column anyway you could re-clone it with a His-tag that is cleavable by an enzyme (TeV, FactorX,... - I am a little out of date about which enzyme is the most favoured on nowadays).
This would add one extra purification step, one with and an withouth His-tag. Since these are complementary, this ought to yield already pretty pure protein.
What buffer did you choose for the Gel filtration - did you have enough salt to reduce interaction with the column? And what exactly do you mean with 'interacts' - does the protein not elute at all? Same question holds for the Ion exchange, what was the gradient you drove?
A "polishing" GF-step at the end of purification is definitely something you want aim at.
Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Mon, 9 Feb 2009, Marek Frischerkase wrote:
Dear all, I´m new in the field of crystallography. So please excuse my first question is off topic. I´m trying to purify a 182 kDa his-tagged bacterial enzyme. The expression in E. coli works quite well. After Ni-NTA the protein isn´t pure enough at all, so I tried several gelfiltration columns, but my enzyme interacts with the carbohydrate matrix, iex didn´t work either. So does anyone have suggestions? Perhaps a 182 kDa protein is too large to crystallize anyway? Cheers, Marek
