Most people use for recombinant proteins, a His tag construct followed by a TEV cleavage site. Then you can use NI NTA purification followed by a possible buffer exchange, TEV cleavage, and then passage through Ni NTA once more to remove TEV and then polish with a sizing column step, that also gets your protein in the right buffer for crystallization. You may need to do ion exchange instead of or in addition to the sizing column. In he case of using ion exchange, I would leave the sizing column as a last step. If it is not recombinant, then the procedure totally depends on what protein you have, and trial and error.

For recombinant proteins, some people use the Gateway system, so they can easily express the protein in multiple systems, if, for example, Ecoli fails, and/or if a different tag is needed. The standard tags are His, GST, and MBP.

Your question surprises me. Purifying for crystallisation is a non trivial procedure, I don't think it can be generalised, it depends on you protein: sequence, solubility, stability, codon usage, internal sites for common enzymes for removing tags, financing, plasmid availability and many others...

if your protein is intracellular and has a tag, then

1] affinity chromatography

2]ion exchange chromatograhpy

3] size exclusion chromatography

4] if is is still dirty, may be HIC chromatography.

If there is no tag

1] MMC chromatography or Chromatofocussing

2] ion exchange chromatography

3] Size exclusion chromatography.

if it is extra cellular, perform TFF concentration followed by the above steps..

there is not really a fast method as far as protein purification is concerned...you take one step at a time and try to achieve purity

good luck

Thank you all for your advices, particularly to Dr Moss. Unfortunately for me, this is my first time in which I'm going to approach to experiments of cristallization...In my scientific life, I have done something different until now, but that is what i love of this work,!

You can contact me anytime on researchgate or not. I don't know too much about expression systems and molecular biology, so maybe others can help you with that. I do know people whom you can contact for just about anything, even for crystallization studies. But here is my email: [email protected]

Thank you so much...In my case, I was lucky, with the expression of my protein complex...but I have a problem with stability of one protein of the complex...I hope to solve as soon as possible the problem to go ahead for crystallization...Any advices to overcome the problem of protein degradation would be appraciate!!!

If the protein is in the complex, and is still unstable, then adding proteinase inhibitors is an option, or passage over columns to remove proteinases is possible. People use a benzamidine, or gelatin chromatography. Changes in the buffer might help too, to keep your protein more compact and less likely to be degraded. Or pH changes depending on what type of proteolytic activity you have. Maybe you can set up a 96 well plate, and change the buffer conditions, and then look for degradation. Changing the hydrophobicity or hydrophilicity of the buffer may help.

The protein may be more stable in the complex, ie it needs other proteins.

But adding proteinase inhibitors is the first solution. Roche diagnostics sells inhibitor cocktail pills. If your protein is not an enzyme, I would add all the PIs you can.

Thank you...at what concentration do you suggest me to use Benzamide as protease inhibitor?

The column is a benzamidine affinity resin. I would have to look to find who sells them.

But if I want simply to use benzamide as protease inhibitor? Do you know the concentration that is recommended?

Thanks

10uM.