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.

Thanks Ryan Wheeler , I’m going to give it a try

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.