I expressed my target protein in pGEXT-4T-2 vector. I got fairly nice amount of protein after purification using GST sepaharose 4B beads. Now when I tried cleaving off thrombin using on-column cleavage (thrombin clean clean cleave kit,Sigma), I don't see anything on my SDS PAGE or western. I can't even see uncleaved protein in elutes. I am trying to repeat the procedure now. Any suggestion on this please?
I incubated my fusion protein with GST sepahrose beads in an eppendorf tube overnight. Then next day, I carry out elution using reduced glutathione. Now my protein is in elution buffer. Next step is cleaving off the tag. Here is what I did: I used sigma clean cleave kit that provides thrombin agarose beads, I incubated my protein with the beads at different time points, took the supernatant and pass it through a column and collected elutes assuming GST and my protein will be seen separately on SDS and western. But unfortunately, I didn't see anything. Then I also collected beads fro the column to see if protein remain bound, but no I didn't see anything. I was hoping to see at least thrombin band or uncleaved fusion protein.
As Cristina mentioned, thrombin might not be very active in your GST-elution buffer, so it's best to dialyse your protein into a buffer where thrombin shows maximum activity.
Your problem, however, is the disappearance of your protein rather than a lack of cleavage. The reason why many people have stopped using thrombin (I'm among them) is that it isn't very specific. It will not just cleave off your tag but also randomly cleave your protein if you leave them together for too long. I have never had my protein samples chopped up by thrombin to the point I couldn't see any protein at all on an SDS PAGE gel (you usually see a number of smaller, smeary bands instead of your full-length protein band), but I guess it's possible that using a large excess of thrombin could completely degrade your protein to the point where the peptides are too small to show up on a gel.
I agree with Antonio - most likely the thrombin is totally degrading your protein. It is a little odd that it also degrades the GST though, which suggests that you are adding rather too much.
I would suggest that you start by trying a small scale digestion with a small sample of your fusion protein. Add some neat thrombin (Sigma's T4648 has always worked for me, or you could just use the equivalent amount of the CleanCleave kit), at maybe 1 U for every 100 ug of your protein. Incubate at 4 C; at various time-points (say, 0 hr, 15 min, 30 min, 1 hr, 2 hr, 4 hr, overnight), take samples and run on a gel to get an idea of how much protein is cleaved, how fast. This should help you determine what amount of protease you need to get sufficient cleavage of your protein without non-specific effects.
Alas, if the protease chops up your protein rather quicker than it releases it from the tag, you will have to switch to a different protease like 3C or TEV.
Thrombin does not seem to an ideal choice of protease for eluting protein from beads. I got Thrombin from GE healthcare and it had poor efficiency as well. This seems to be a common problem. Best is to clone into the vector with precission protease. Even there the cleavage of the tag might be highly efficient but your protein of interest might stick to the beads.
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