"Leaky" stepwise elution of active protein from Ion-Exchange columns - (Apr/01/2016 )

Hello,

I am working on purification of a cytotoxin from spent growth media (cell free supernatant) of a bacterial species. 

After AmmSulphate precipitation and dialysis; I have been using the HiTrap IEX selection kits with weak/strong cation and anion exchange resins to find the best support to begin the purification.

Using the estimated PI of the protein of interest I have been using both cation/anion resins to bind the protein of interest (>1pH buffer either -/+ ) and found the majority of "activity" binds to the column (not in flow through)

However, when I use a stepwise NaCl elution (0.1M-0.2M-0.4M and 1M) to fractionate out the proteins, rather than coming off at one fixed salt concentration in a defined peak (activity peak, not A280), the active protein seems to come off a little in each salt concentration.

(I have attached an image to represent this pretty reproducible elution profile)

Does anyone have any idea why this may be happening? It occurs in both strong and weak cation and anion exchange resins.

If I assume that the cytotoxic protein is one specific protein (and not multiple, which I have no reason to suspect) what would cause small amounts of this protein to come off in drips and drabs rather than a defined elution peak at 1 salt concentration?

Thank you in advance for any suggestions.

Chris

Some things to think about:

1. Have you tried a linear gradient elution? It is not uncommon during a linear gradient (as opposed to step-wise gradient) for a protein to elute within a range of salt concentrations, eg 200-300 mM.

2. Is it possible there are multiple (iso)forms of your protein? Perhaps differently proteolyzed pieces (all of which are active to some extent)? Or different oligomerization states? Or different post-translational modifications (e.g. disulfide bonds, crosslinks, acetylation, etc)? Should there be any co-factors bound to it and only some of your protein has this co-factor, which could change shape, etc of your protein? Basically, If there is any reason to believe your one specific protein comes in multiple flavors, each flavor may elute off the ion exchange at different salt concentrations.

3. Consider the reverse setup. Take your amm. so4 and resuspend in high salt, and don't dialyze. Apply to hydrophobic column (eg phenyl sepharose) and elute with gradient of lowering salt concentrations. You may get a single elution activity peak from that.

Thank you for replying Labtastic, I had stopped checking the thread as no one had replied.

We had attempted a linear gradient initially and saw a similar tailing across a large range of fractions, so turned to the stepwise gradient instead.

It is entirely possible that the protein is cleaved or modified at different locations, and I do like your idea of multiple isoforms existing in the cell-free supernatant, each with "activity" but different physical characteristics. Thank you for the suggestion

I will also look into HIC as you suggested, that may be a better start in the purification scheme after amm-sulphate. It will take some time but I will update for the sake of closure when I find out.

Thanks!!

Chris