My doubt is about the considerations on the purification of a recombinant protein using hys-tag, is it forbidden by the FDA to do so? Can I leave the tag or I must remove it previous to clinical trials? Thanks
I do not know of the regulation on his-tags. It may be immunogenic though if you inject it into a human body.
You could try using TEV protease to cleave the tag off, it was said to work well. TEV recognition site can be altered, so that your protein can keep it's native aa sequence.
If it is an ER protein, you can add a ER-protease cleavage site that cuts off the tag without leaving residues behind. You would have to express your proteins in eukaryotes then, not bacteria obviously
An important consideration if a protein is to be used as a therapeutic in people is that it can be prepared reproducibly. If you are going to cleave off the tag, you have to able to do it to the same extent every time. Sometimes the cleavage reactions don't go to completion, which makes them hard to reproduce. Also, there is sometimes a low yield of protein from the cleavage and protease removal step, which could drive up the cost of production. So it might be better, from a process point of view, to leave the tag on, if it is allowed.
You could remove the tag by running over a nickel column again... The proteins that still have the tag on will remain bound to the column, the cleaved proteins flow through
Thanks Adam, I might do so. Andreas, thanks first, and that's exactly what we are planning to do to remove the remaining tags in the buffer. Another thing: is there any supplier of industrial quantities of His-trap or any Ni NTA resin? The problem is this resin represents around 27% of the whole compound investment in a year. Thanks again.
If cost is an issue, why don't you switch to IDA (way cheaper than NTA)? You will have to spend time optimizing the run, though.
I actually don't have a clue, but I doubt the FDA specifically forbids the use of Ni-NTA (although I'm sure they will require a demonstration that all Ni leaching from the resin has been removed). Also, if the protein is intended to be administered repeatedly, the (low) immunogenicity of the His tag will most likely result in the appearance of anti protein antibodies with time, and regulators will probably latch onto that.
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