I recently attempted to cleave the tag of my protein of interest using Sigma's recombinant enterokinase E4906. However following negative purification, all the protein still eluted in the elution fraction, and there was no apparent cleavage at all. I carried out the cleavage over night at 5 degrees. The pH was between 7-8, and the mixture was supplemented with 2mM CaCl2. I had just dialysed the protein sample so there should not have been high concentrations of imidizole, salt or reducing agents. Perhaps the activity is too low at 5 degrees? I am going to try cleavage at RT just to see if it works at all but I am concerned about protein degradation. Does any one have any experience of tag cleavage with EK?
Concentrate 4º sample (with EK protease) and cleave at room temp.
That is a pretty active protease, so if it doesn't work after concentration, you may have gotten a bad batch of EK.
I use enterokinase to remove the GST tag from a protein while still bound to glutathione agarose, much as you describe. For my protein, the on-column cleavage is not very efficient, so what I have to do is incubate the resin with EK overnight at 4C, and in the morning collect the FT and add fresh buffer and EK and incubate again. I do this up to four times with a single batch. At the end I pool the FTs and concentrate. I must do it this way because EK digests the GST-protein completely if the digest is done to the purified fusion protein. The main differences I see in what I do and what you are doing is that I use EKmax from Invitrogen and the digest buffer I use is 50 mM Tris HCl pH8.0, 1 mM CaCl2 and 0.01% Tween 20. The Tween really helps in the case of my protein.
Hope this helps, and good luck!
Thank you both for the advice. I'm just running the gel from my previous cleavage overnight at RT. If that doesn't work perhaps I will try as you suggested. The manual for the EK I have does actually suggest to use 1% tween, but we didn't have any at the time and I didn't think it would make such a big difference..perhaps that's the problem.
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