Need your kind help/suggestions:
I am trying to purify two 6X-His tagged proteins/enzymes (isoforms) in native form and already have one of those in hand (in very good quantities). Although my protocol worked with one form, the other appears to simply not bind the Ni—NTA beads (from Qiagen). The induction conditions, purification protocol, quantities expressed in soluble and insoluble fractions are almost similar. These protein were already expressed/purified once (long ago) in the lab by a colleague and I am using the same clone (though I used my own transformant E. coli) so there is almost no chance of clone defects. Also, I am getting the expression in my case with reasonable amount in soluble fraction (though remarkably lesser compared to insoluble quantity).
These are few relevant specifications:
1) I used Phosphate buffer (no imidazole in binding)
2) Wash buffer with 20mM Imidazole
3) Elution with 100mM Imidazole.
4) Binding time 4-5 h
I am thinking of following solutions:
1) Use another transformant (another colony); however, the transformant I used expresses the protein of desired molecular weight.
2) Check the accessibility of His-tag through binding under denaturing conditions (although it was earlier purified in native form suggesting the positive accessibility of tag). This may give an answer but eventually I need my protein/enzyme in native form.
3) Increase the binding time to overnight, though in earlier attempts 5 h worked perfectly fine.
4) Add 1-5 mM imidazole in binding to reduce the non-specific binding (as I see many non-specific protein in the wash fractions and elute as well) and allow binding of my protein.
I need the protein urgently and wish to cut short the troubleshooting steps and/or limit it to some modifications in the purification protocol. If anyone had a similar experience and have some suggestions, please share.
Failure of His-tagged proteins to bind to Ni-NTA frequently is caused by the protein forming soluble aggregates. The fact that a large amount of your protein is in the insoluble fraction already hints at a folding problem: If you are not already doing so, try to express your protein at lower temperature.
Thanks for your suggestion Annemarie. I have expressed my protein at 16 degrees O/N.
I have following suggestions:
1. Add 10 mM imidazole in the binding buffer. It will help you. Before loading on the column, recharge the column with nickel.
2.. I use 300 mM imidazole during elution.
One question, for how much time you are expressing the protein after induction with IPTG?
Regards,
Amit
Hi Amit, Thanks for your suggestion.
I am using Ni-NTA Agarose beads from Qiagen (fresh each time).
I will try another round with 10 mM imidazole in my binding buffer for sure.
I have used the gateway Vector pDEST17, where the inducer is L-Arabinose. I am inducing the expression with 0.01% L-Arabinose for O/N.
Hi Ratnesh,
Just to be sure:
How do you know your protein is not binding?
The obvious way is to see your protein of interest in the flow through of the NiNTA, but as you did not mention that, I still wonder if your problem maybe an elution one (I can think that your protein is degraded during the incubation and/or 100 mM is not enough for whatever reason...)
If it really cannot bind, well, I'm also afraid of a specific near-terminal digestion that cleave your tag away... Do you add any enzyme inhibitors? Can you lower the incubation time? I used 1h for various constructs without any problem... (you can even just make it flow through directly without incubation)
Best wishes,
Guillaume
Hi Guillaume,
Thanks for your time and comments.
The quantity of protein in the lysate supernatant (before binding) and the unbound fraction is almost similar (visually on Gel). I add 1mM PMSF as a protease inhibitor.
If the protein is still there in the unbound fraction after 5 h of incubation with beads, do you think reducing the incubation time may help?
Regards,
Whether or not your His-tag is cleaved can be assed by a western blot of your crude extract, using anti-His tag antibodies commercially available. The same type of blot will show you whether the concentration of your protein-of-interest is significantly lower in the flow-through of your column (if the protein is retained) or nearly the same as in the crude extract (if it is not retained in the column. Taking a small aliquot of beads from the top of the washed column before elution and boil them in SDS loading buffer to see whether the protein is not eluted because it precipitates on the column,
and also analyzed the eluted fractions to see whether the protein is washed off in such a broad smear that you cannot see a peak. This should help to discern the cause of the problem.
Hi Ratnesh,
For your question, if you don't have tag cleavage I would say reducing the incubation time will not change anything...
I think now you have enough options to try out in order to understand what is going wrongly (protein degradation or soluble aggregates)... let us know about your progress
Best wishes,
Guillaume
Hi,
I just recalled that the stock of protease inhibitor (PMSF) I used was prepared in DMSO. Also, I noticed it precipitating out while adding to my cold lysis buffer (may be as DMSO freezes below 18 degrees).
My question is if a near terminal his tag proteolytic cleavage is possible at 4 degrees? I carried out all my steps on ice and the incubation with beads was carried out inside 4 degree room for 4 h (which is a pretty long time).
Also, as same was done for the other protein and it was well purified, does it varies from protein to protein.
As I am still not so experienced with protein purification work, could you please suggest if proteolytic degradation is a big hurdle and it specifically cleaves out the His-Tag, as the unbound fraction still have my protein at a desired weight range (on Gel).
Just for some more information, I do see a very weak interaction with beads, and some protein (very less) in wash fractions and elute as well. However, most of the protein is in unbound fraction.