I am working on recombinant production of a peptide and thioredoxin as a fusion partner in E. coli. My fusion protein has also 6 histidine tag. This fusion protein is produced as inclusion body. When I use Nickel columns for purifying the histidine-tagged protein, I face several problems. When I use Ni-TED column, the protein doesn’t bind to the column and all proteins are in the flow through (!!!). When using Ni-NTA and Ni-IDA column, I see some non-specific bands in elution. I have done some modifications in solubilization and washing procedures (e.g. including imidazole (10-20 mM) in binding and washing solutions, increasing NaCl concentration, including β-Mercaptoethanol (30 mM) and Triton x-100 (1-2%) in washing solution, increasing wash volume, …), but the problems still exist. I have analyzed the sequence to check if the fusion protein and the histidine tag are in the frame. The sequence is in the frame. So, I am looking for alternate methods for purifying the fusion protein. Can anyone help me in this matter? Which methods could be suitable in my case?
What is the molecular size of your target protein in its desired native form? Did you run the cell lysate on SDS-PAGE? What are the molecular sizes of the major contaminant proteins from the cell lysates? I am asking these questions to suggest centrifugal membrane filtration with appropriate molecular weight cut off (MWCO) to capture your proteins.
Are the tag proteins essential for the desired activity of your target protein? If you are able to cleave the tag proteins, you can try selective salting-out precipitation to separate your proteins from soluble contaminant proteins. For obtaining highly purified proteins, you may need to use SEC for post precipitation polishing of your proteins from soluble aggregates and other misfolded proteins and contaminant proteins.
You mentioned that the Ni-NTA and Ni-IDA have non-specific bands during the elution. Is your protein of interest included in the elution? You should not expect that your recombinant protein will be absolutely pure from a single step of affinity chromatography. Do you have any evidence that the His-tagged fusion protein binds the resin ever (ie the plasmid was used in a previous purification project with great success or this is a new plasmid that you generated)? I am pretty sure this won't work, but have you tried Talon? You also didn't mention pH... Make sure that your pH is 7.5-8.5. Sometimes a fusion protein or recombinant protein interacts with the histidine tag and prevents the interaction of the tag with the affinity resin.
With that said, you can always purify ANY protein using old-school methods such as salting-out with ammonium sulfate, Hydrophobic Interaction Chromatography, Ion Exchange Chromatography, and Size-Exclusion Chromatography. I would focus on Anion-Exchange chromatography with a quaternary amine ligand using Tris at pH 7.5 and eluting with NaCl from 0mM to ~600 mM as this purification technique is mentioned in the literature.
As a general purification scheme of non-tagged proteins, resuspend inclusion bodies in NaPO4 buffer, add 80% saturation of ammonium sulfate, let the proteins precipitate at 4C, centrifuge, resuspend in NaPO4 and 1.5M Ammonium sulfate, inject over HIC (I prefer phenyl ligand), elute with a decreasing ammonium sulfate concentration to 0M, desalt sample and buffer exchange into Tris or HEPES with minimal salt, inject over Anion-Exchange with Q ligand, elute and then polish with SEC.
Purifying his-tagged proteins requires at least two chromatographic steps. I prefer imac and then iex. As Carter says imac is a pseudo-affinity technique, thus you can always expect to adsorb other proteins, no matter what you do to increase specificity.
Anyhow, the first thing you should be doing is changing your protocol such that your protein is expressed in its native form and not as an inclusion body.
Thank you all.
Dear Alemu, the size of fusion protein is around 20 KDa. Actually the fusion partner is not essential for the peptide bioactivity. I use the fusion partner because the peptide itself is toxic for the host (E. coli). After purification, I have to cleave the fusion protein.
Are you going to use SEC after tag cleavage for separation of your peptide from the tag protein? Is your peptide more hydrophobic?
I would like to recommend RP-HPLC method (please see file; Lysozyme by RP-HPLC) and/or HPLC-Surf-SEC method (please see file; SEC column 300Åsilica). Please, protect cysteine-residues by pyridyl-ethylation method (PE method; please see file; PE derivative) in RP-HPLC. Please use 100 Åsilica for Mr 20,000 protein in HPLC-Surf-SEC.
Did I miss some step here?
"My fusion protein has also 6 histidine tag. This fusion protein is produced as inclusion body. "
So how do you get it out of inclusion bodies? How about changing expression conditions (use M9 medium, add less inducer, lower growth temperature to lower expression rate, add NaCl to cells to induce chaperone expression) in order to increase target protein solubility?
Yoram
I partially agree with their views, such as do more steps to purify. Like combine affinity, ion exchange, gel separation.
But I do think the really important thing is inclusion body. It is natural phenomenon, since the bacterial do not want to express proteins for others. I recommend you to recovery the inclusion body first, and then purify the fusion protein with Ni followed by ion exchange , and the gel isolation.
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