I bind my fused GST-protein to glutathion beads and I cut with Prescission at pH 7.5 to eluate overnight in batch method, sometimes my protein (always the same clone) remains attached to beads after Prescission digestion. It is a bad lot of Prescission (I tried two new lots now ans it is still the same)?, I keep my beads with the fusion protein in the fridge (+4ºC), does anyone have an idea to facilitate the protein release?.
Now, I cut the protein in two small fragments with pI 4.6 and 7.8 both fused to GST, I will expect not to have pH problems in elution, I need to change my elution conditions?
Hi there,
Have you checked the digest is actually efficient in the batch context by loading beads on SDS/PAGE? If protease is fully active on immobilized fusion then you might be observing the drawback of using solubilizing protein partners: the fusion behaves OK because tag is improving the protein solubility of the protein of interest. But as soon as you cleave the fusion, your protein behaves badly and precipitates or aggregates onto the beads because GST is no longer stabilizing your protein.
An other explanation is that although efficient cleavage GST and your protein are still closely interacting. If so increasing elution stringency (in terms of ionic strength for instance) should improve elution. To check if it is the case, elution with GSH should elute both your protein and GST.
Hello Dominique,
Thanks for your answer, yes I test the beads and the fusion protein is still in the beads with a new Prescission, so as I used always the same clone from the same glycerolate I suppose it is not my construction (already sequenced many times) and I was doing the same protocol several times, I think I have troubles now with the enzyme. I keep my beads in the fridge waiting for a new lot. I attached a picture of coomassie.
So the digest is always uncomplete: 2 possibilities : protease activity is low and/or cleavage site accessibility is limited when fusion is immobilized on beads. To check this, try to digest the fusion in solution. If OK in solution then the problem is accessibility, if not then it's a protease problem.
What I don't get from your gel is that fusion, GST and your digested protein are all present in elutions... Normally GST and fusion should remain on beads.
Maybe it will be better to change my vector and use another tag, histidine alone or with other one, which pET will be more useful?
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