I have came across the protocols that define the cleavage of His-tag from His-tagged fusion proteins with TEV protease using purified protein samples.
I was wondering if we can apply the protocol for a transfected cell lysate sample of the fusion protein, keeping in mind the reaction conditions. I want to run a pilot cleavage experiment without purifying the protein. If anyone knows about it kindly let me know.
Hi there,
I've never performed such experiment but in theory it should work as TEV is very specific for its cleavage site so your POI should be its preferential substrate even in a crude extract... Provided there is no proteolytic activity which could degrade the TEV protease.
But what's the point of getting rid of an affinity tag prior affinity purification?
Thanks Dominique Liger for the answer. I just want to check that cleavage works before going for a bulk production of my protein of interest.
If I were you I would perform a mini purification first to prove that the affinity step is efficient... And then check TEV activity on the purified fraction ... Or use the TEV activity to elute the protein from NiNTA...
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