For my experiment I have immobilised a his tagged protein on Dynabeads his-tag isolation and pulldown magnetic beads that will be used to pull out specific cells from PBMCs. I need to enzymatically cleave the tag off to release the protein and the cells from the beads. From my readings I've learnt that I can include TEV recognition sequence in my recombinant his-tagged protein and subsequently use TEV protease to cleave the tag off. To illustrate, the sequence would be HHHHHH - ENLYFQG(TEV recognition site) - protein sequence.
The problem is, most TEV enzymes on the market are themselves his-tagged, which means that upon addition to the beads, the enzyme will be immobilised on the beads. Finally, my QUESTIONS:
- is there another protease that would be better suited to the job (must work under physiological conditions and not have a his-tag)?
- if my protein and TEV are both his-tagged at the N terminus meaning the enzyme and the recognition sequence are in close proximity, could the reaction still proceed when both are immobilised on the beads?
Hi,
I think a simpler approach would be:
1. Elute your target protein from the magnetic beads
2. Dialyze the sample to a buffer without imidazole
3. Cleave the his-tag off of the target protein with TEV
4. Use Ni-NTA magnetic beads to remove the TEV and cleaved his-tags
Good luck,
Ryan
Thanks for your suggestion, Ryan Wheeler. The problem is I can’t elute the protein off the beads as by this stage the protein is attached to cells. The low pH of the elution buffer pops the cells which is highly undesirable. The whole purpose of the experiment is to recover the cells rather than the his-tagged protein which is used as a bait.
Hello,
You can elute the cells at a neutral pH if you use imidazole. I do a similar experiment with E. coli where I use magnetic beads to isolate them. I elute 2 times with 50 uL of 500 mM imidazole in tris buffer, pH 7.4, and the cells are viable after.
I seen this paper where they used TEV to eluted his-tagged proteins on beads. Article An Open Library of Human Kinase Domain Constructs for Automa...
But I'm not 100% sure if they used a HIS-tagged TEV, but from reading the paper it seems like they used TEV without HIS tag because they had TEV contaminants afterwards. Regardless, it is relatively straightforward to purify TEV without HIS-tag on your own, and I highly recommend doing so.
Either way, I've read that TEV is more high yielding when cleaving in solution than in solid phase.
If all else fails, thrombin protease is also an attractive option.
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