I have a construct that goes (N to C terminus) 14x His tag - SUMOstar cleavage tag - Protein of Interest (POI).
The initial idea was to elute using on-column cleavage of fusion protein, but this resulted in very low cleavage, even though my lysate-only controls showed significant cleavage (even in the presence of a protease inhibitor cocktail). I also couldn't release the fusion protein from the column with a 250 mM imidazole wash to perform cleavage off-column. The only thing that removed bulk protein from the column was to strip it with 100 mM EDTA, which is obviously not good for the resin. I think a big part of the problem is when the fusion protein binds to the column, the protease isn't able to reach the cleavage site very well.
I was wondering if using a cobalt resin (Takara TALON if that matters) rather than a nickel resin would allow at least some elution of the fusion protein with high imidazole washes, since cobalt resins are supposed to bind his tags less tightly than nickel resins.
Dear Alexandra
EDTA stripping is not a problem for the resin. I routinelly perfom it, to strip the nickel and regenerate the coloumns after usage. It depends if EDTA is a problem for your protein or for the following steps.
Probably i'm wrong but it sound strange to me that presence of 14 histidine is able to link your protein soo stong to the Ni-NTA resin that you are not able to elute it with 250mM imidazole? Did you tried to increase it at 300mM or 500mM?
I have purified several time protein with 10 histidine and those elute very well with 300mM imidazole. It seems to strange that 4 more histidines make a so huge difference.
Did you succed to digest the protein after stripping with EDTA? Because sometimes the problem with protein cleavage can be due to protein aggregation and i have the doubt that the missed elution could be also due to the fact that your protein is not aggregated/precipitated in column.
But this is just an hypothesis
ciao
Manuele
Hi Alexandra Johnson . I totally agree with Manuele Martinelli . Adding 14 His to a protein terminus is more likely to screw up protein structure and solubility and not help for ionic based IMAC purification.
Imagine adding 14 Arg to a protein terminus and expecting it to behave normally in conventional anionic chromatography.
This is the sequence of Histidine Rich Protein 2 from Plasmodium falciparum:
MAKNAKGLNLNKRLLHETQAHVDDAHHAHHVADAHHAHHVADAHHAHHVADAHHAHHVADAHHAHHVADAHHAHHAADAHHAHHAADAHHAHHAADAHHATDAHHAADAHHAADAHHAHHAADAHHAHHAADAHHAHHAADAHHAHHASDAHHAADAHHAAYAHHAHHAADAHHAADAHHAADAHHAADAHHATDAHHAHHAADAHHAADAHHAADAHHAAAHHATDAHHAAAHHEAATHCLRH
It is 37% Histidine by composition. No recombinantly added polyhistidine tag is required. The protein literally is a HIS-tag. Laugh (HAHAHA) but it is a real protein despite the fact that it looks made up: https://www.uniprot.org/uniprot/A9XLQ2
It literally has a biological function and the protein can be recombinantly expressed in E.coli and captured on a Ni-NTA resin and elutes with 300 mM Imidazole. The authors tried an imidazole gradient from 200 mM to 1000 mM and that is what they found (Lynn, A., Chandra, S., Malhotra, P., Chauhan, V.S., 1999).
You are not using enough imidazole. Don't give up so soon without even trying.
EDTA is not a problem for Ni-NTA resins because the resins can be regenerated with Nickel Chloride or Nickel Sulfate. EDTA will be a problem if your enzyme requires divalent metals for catalysis. Full inhibition.
You can dialyze the imidazole or EDTA out of your protein by dialyzing the protein in a membrane in a larger volume of buffer that doesn't contain those things.
Sources:
Lynn, A., Chandra, S., Malhotra, P., Chauhan, V.S., 1999. Heme binding and polymerization by Plasmodium falciparum histidine rich protein II: influence of pH on activity and conformation. FEBS Letters 5.
https://www.uniprot.org/uniprot/A9XLQ2
Adron Ung. So you are just equating a naturally occurring His rich protein to engineered proteins with 14 His on their tails with respect to IMAC binding and nothing else, right?
Steingrimur Stefansson , yes I am. That protein is intrinsically disordered by circular dichroism until it binds its ligands so its like a big HIS-tag. A His-tag could conceivable interfere with protein expression but when the polyhistidine tag is cleaved from a fusion protein, the protein should be fine.
Nickel-NTA affinity cheomatrography for polyhistidine tagged proteins is wonderful. Unfolded proteins with a polyhistidine tag will bind to the resin, and the captured target protein can be refolded on the resin. Other affinity purification methods require folded proteins, but Ni-NTA chromatography does not.
Adron Ung . I was about to answer you before you added chemistry. I detected a hint of creationism in your....never mind.
I have done CD. Did a lot of it for my PhD. It is a crap tool except when proteins go bonkers when denatured.
You said (captured/copied text):
"Nickel-NTA affinity cheomatrography for polyhistidine tagged proteins is wonderful. Unfolded proteins with a polyhistidine tag will bind to the resin, and the captured target protein can be refolded on the resin. Other affinity purification methods require folded proteins, but Ni-NTA chromatography does not."
Um..please tell me you are just joshin' me. Ni-NTA is no better than Zn-NTA, Co-NTA, etc, etc.
I never said anything about creationism so not sure where you are getting that from. I mentioned Ni-NTA because I have experience using it and it is cheap and works well to regenerate the resins with Nickel Chloride.
Perhaps arguing doesn’t achieve anything. Thank you for your feedback.
As far as affinity chromatography goes, capture of His-tagged proteins is much more convenient than GST tagged proteins as the latter requires a folded protein to bind to the Glutathione resin.
All IMAC resins are cheap and work with most transition metals.
As far as protein purification goes, most of what is labeled "affinity chromatography" (IMAC and Hep seph) is just glorified anion/cation exchange resins.
Real affinity chromatography is when you get into pseudo substrates for enzymes, like benzamidine sepharose for trypsin-like enzymes and immobilized Abs..
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