It depends on what purpose you want to purifiy your protein for. Ususally, if your protein is well produced and if the Ni affinity chromatography is performed correctly (i.e. adding ~ 20 mM imidazole in the wash buffer to prevent non-specific binding of unwanted proteins), the protein should be sufficiently pure for enzymology tests. However, if you intend to try crystallization, a polishing step is recommended. Gel filtration is generally chosen for this, as it will remove potential high molecular weight aggregates, which will hamper crystallization.

Another point is removal of the Ni affinity tag. This may be critical for crystallization, and also for enzymology, as the tag may affect the properties of the enzyme. Thus, it is preferable to chose an expression vector that provides a site for a processing enzyme such as Tev protease in the encoded polypeptide.

Yes I'm preparing my protein for crystallisation. Thank you Pierre for the answer.

Hi there,

One step purification is generally not enough as whatever the purification criteria applied you wouldn't get a pure and homogenous protein (which is generally necessary for downstream applications). For instance affinity chromatography is highly selective concerning the content of the target protein but doesn't make any difference between subclasses of the protein population (monomer, dimer, oligomer, agregate, folded, unfolded). That's why at least a SEC step is required to try to resolve these different populations.

Thanks Dominique

Comments following are ones which were not explicitly expressed above:

1. These proteins of interest are either ezymes, transcrition factors ...... on and on. This means they are ligands too for other proteins, cofactors, and/or substrates.

2. Small Molecules like cofactors or substrates may co-purify along with the hist-tag. Not visible on a SDS PAGE

3. For crystallization, you'll need very pure protein with which you may need to carefully add back chaperones for efficent crystallization.

4. Check the websites below for hints in crystallization:

https://www.creative-biostructure.com/crystallization-chaperone-strategies-21.htm

Methods 55(4):293-302 · August 2011 (Check these people out)

To Your Success - Look forward to following your future success!

Dan

Thank you Dan

Presumably you are purifying it for structural biology studies like NMR or X-ray crystallography. If that is the case, then the first pass over Nickel affinity is to separate your His-tagged protein from the rest of the proteins in the lysate. You should use sufficiently long column wash and monitor the column effluence by UV-Vis to make sure you get all the other protein separated. The SDS Page is to check purity at each step but doesn’t tell the whole story. I’ve seen co-eluting conformers of identical size under a single SDS page peak that are only resolved by a high res Size-exclusion/gel filtration step. Next you can cleave your protein with a protease and either run it back over the Nickel colum, this time to bind uncleaved tagged protein which guess what, will likely co-migrate under SDS-Page since the tag is so small that SDS-Page often cannot resolve cleaved from uncleaved. A final polishing step of ion exchange guarantees your purity, so it depends what you are trying to do with it. Remember that protein is an ensemble of different conforme only maintained by strict control of ionic strength, buffer, pH, temperature and any deviation will effect the protein etc. I hope this clarifies why you need so many steps. You are asking the right questions, but there is no single purification strategy that will work for all proteins. You must consider them individually, and if you are using the protein for structural biology- polished purity is the name of the game and a single step purification is a fantasy

I am very sad to express my opinion. Combination of Ni- or Co-affinity gel and His-tag-protein is surely not good (my unpublished experience). Affiniy gel using metal is not effective since many metal binding proteins are present (proteases are all have affinity to metal or have metal as cofactors, and transcription factors are Zn-proteins). Further, many Affinity gels also have strong affinity to the hydrophobic membrane proteins (my unpublished observation).

I have previously affinity purified the brain lipoamidase (hydrophobic membrane glycoprotein) at a final step by immobilized Arg-Phe-amide gel (please see file; LIP affinity purification). Interestingly, immobilized lipoic acid (thioctic acid) gel is not effective. Brain lipoamidase is a multiple hydrolase (please see file; multiple hydrolase LIP) having affinity also to the neuropeptide of Arg-Phe-amide. Pig-brain lipoamidase has Km to Lipoamide at 502 μM, but has Km to Arg-Phe-amide at 208 μM, respectively (please see file; Multiple hydrolase LIP).

Sadly, biotin-binding protein of avidin unexpectedly turned out to be the multiple binding-protein for lipoic acid/biotin/amino acids binding protein.

Therefore, purification of lipoamidase has been performed using various steps (please see file; Purify Brain Lipoamidase).

I am now considering that the RP-HPLC method is most powerful and reliable method to purify proteins (please see file; Lysozyme by RP-HPLC).