Asked 25th Jul, 2012
How to remove a GST tag from my recombinant protein?
I have cloned and expressed my protein in BL21DE3 cells using pGEX-4T2 vector and have succesfully purified the recombinant protein using an affinity column (glutathione). When I tried cleaving the GST tag with the RECOMT thrombin cleavage kit (sigma), I didn´t see any cleavage of the tag from the protein when looking at the gel. I have been facing this problem for some time and have not yet been able to obtain protein without the tag. I have carried out the reaction at around 25 degrees, as directed by the kit, for 24 hrs but with no result.
Can anyone please tell me if you have used another technique or kit for performing GST tag cleavage?
Most recent answer
All Answers (11)
1) Read the GST-fusion manual available from GE healthcare and make sure your sample is not contaminated with protease inhibitors. 2) Make sure you have the right protease. 3) If you are trying an on column cleavage, try eluting the protein with GSH, dialyse away the GSH then add the protease. Note that factor Xa is inactivated by GSH as it reduces an essential disulphide bond. Similar story for thrombin and DTT. High activity Human thrombin from calbiochem is excellent, cheep bovine thrombin is not so good.
@Jason Crack I didnot used any protease inhibitor during any of the isolation and purification steps and have also dialyzed the eluted protein to remove reduced glutathione.
@Ties Latendorf the protease recognition site is present in the cloned vector and was confirmed through sequencing.
Not only us but other labs in our department are also facing the same problem. Right now we are thinking of ordering free thrombin and use it for the tag removal .
Ankana, I had the same problem with the RECOMT thrombin and changed to thrombin from Amersham and I worked really well. What size is your protein? Remember that for you to see GST clearly it will be better if you run a 15% SDS-PAGE. Hope this information is useful!
First check the protease recognition sequence on your total protein,then check the selected protease is suitable or not, then check what are the co factors are necessary for our protease activity. and try different concentrations of protease & protein reaction mixture.
you must take care of temperature and pH during reaction.
Dependent on the properties of the fusion partner, the cleavage site sometimes is structurally covered by the fusion partner and thus not acccesible for the protase under physiological conditions. Including non-denaturing concetrations of Urea (0.5-1 M) might help to get cleavage work. Another point is that GST often promotes solubility of the fusion partner, but without GST the protein starts to aggregate and forms a mixture of GST and fusion partner aggregtes which can be resistant even to high levels of SDS and b-ME leading to an apparent molecular weight of the full length protein. Good luck !
I agree with Michael Muehle, we also faced similar problem, we used two pGEX4T1 and 4T2 and faced same problem even after cheking every thing, sequence, extensive dialysis, buffer, temperature, incubation time. The band of Thrombin treated protein and untreated samples show up at same point on SDS. I strongly believe there is a problem with PGEX vectors in terms of accessibility of thrombin to its cleavage site.
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