Hi Marek, I agree with Tim. 182kDa is certainly not too large for crystallization.
As for purification ..... Have you tried different buffer conditions for your Ni-NTA purification ? Adjusting the pH or salt component may improve the purification sufficiently. You could try changing the salt completely (NaBr rather than NaCl seems to help for some of my proteins) or playing with concentration. I have had success with buffers containing 5mM up to 500mM salts. Also, Tris might not be the best buffer to use with Ni columns if weak binding is an issue, so try HEPES or something else. Do you include some (5-10mM) imidazole in your lysis buffer ? This would prevent some of the contaminants binding to your column initially which may affect how your protein is interacting with the matrix. You could try charging your metal affinity column with something other than Ni. Strip off the Ni with EDTA and recharge with Co2+ or Zn2+, for example. As for ion exchange, again adjusting pH can give good results. Also, you can try (from GE) Heparin or Blue columns. Even if your protein does not bind, perhaps your contaminants will, and their 5ml HiTrap columns certainly won't break the bank. Gel Filtration would be a good polishing step, as Tim suggests, but I guess if you are losing all your prep on that column, and you can achieve a pure homogeneous sample by the other methods, then its not necessarily essential. Or worst case, re-clone with a different tag, or no tag at all. We recently worked with a no-tag construct which purified (~95%) on a single Q sepharose column. But maybe we were just plain lucky ! All the best. Magnus Dr. Magnus S. Alphey Wellcome Trust Biocentre College of Life Sciences University of Dundee Dundee DD1 5EH Tel: +44 1382 385762 Fax: +44 1382 345764 The University of Dundee is a registered Scottish charity, No: SC015096
