I am trying to purify two recombinant proteins that I overexpressed from E. coli BL21 using the pET28a vector. The proteins should only have his-tags on their N terminus since I kept the recombinant genes stop codon, which should stop translation before the C terminus his-tag can be expressed.
When I load the lysate into my IMAC column my recombinant protein appears in the flowthrough while a protein that appears to be endogenous (around 45kDa) in BL21 is appears in my elutions, while the target protein doesn't appear at all. Other proteins I obtained in the same manner were purified without this issue.
I have checked the pH of my buffers and adjusted them to 7-8. I am worried the his tags may be embedded in the protein. While denaturation is an option, I will also have to renature the protein for the next step of my experiment.
Has anyone had any similar experiences? I would appreciate any advice you may have.
Kindly see the binding of your tagged peptides at C or N terminal either they bind to C or N terminal of the proteins because you have look for the binding capacity first, if they have binding capacity then only you can tagged with the specific protein. The static binding capacity (SBC, also called total protein capacity) is normally measured in batch mode in a beaker. SBC is usually reported as the maximum amount of protein bound to a chromatography resin at given solvent and protein concentration conditions.
The easiest and fastest way is to determine the adsorption isotherm, any book dealing with adsorption will guide you on how to do it. You can also measure capacity using frontal analysis but it requires a lot of protein if capacity is high.
Hi there,
Did you make sure of the presence of the tag on the protein in the lysate? (by WB with anti His antibody)
You may try to run imac in mild denaturating conditions to make any hidden tag accessible.
Otherwise try with the tag at the other extremity.
How did you confirm your protein in flowthrough fraction? As Dominique Liger indicated, is your tag reliably constructed and validated through immunoblotting?
Possibly your tag as you suggested is at the inner side of the folded structure and it is hindered. Refolding is overwhelming, unfortunately. If you don't have a chance to switch the tag, or at least its position you may test Cobalt NTA instead of Ni-NTA (assuming your resin is Nickel-based). Cobalt has higher affinity and using it in batch mode instead of column chromatography may improve the binding. Omit from denaturation as possible as you can...
As have been said, confirm that the his-tag is there. If it is, it may be embedded in the structure and not available for binding as you say. You then have to denature the protein, for example, in up to 8 M urea, load it on the Ni-resin which is in the same buffer. The his-tagged protein is binding and you can do a refolding on the column with the protein attached by a long linear gradient (20-30 CV) going from 8 M to 0 M urea and then do the elution of the protein. It can sometimes be easier to do the refolding on the column, but usually you loose yield.
Another thing have you tried to just slowe down the flow rate during loading?
Thank you for your answers, I haven't been able to check for the histidine tag since I don't have anti-His antibodies, however I will definitely try a western blot to ensure the tag is there. I also tried purification under denaturing conditions (8M), and the protein still appeared in the flowthrough. I plan on cloning the gene with a His-tag on the C-terminus as well.
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