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

Deleted profile
I think you should first check the vector to see what cleavage site is between your protein and GST tag.

All Answers (11)

25th Jul, 2012
Jason Crack
University of East Anglia
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.
1 Recommendation
25th Jul, 2012
Ties Latendorf
Laboratory for Clinical Research
First check the activity of the protease by a control-protein. Second check the protease recognition sequence on ProteinLevel by MassSpectrometry or by sequencing the pGEX-4T2 vector with the cloned insert on DNA-level.
26th Jul, 2012
Ankana Kakoti
Salk Institute for Biological Studies
@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 .
26th Jul, 2012
Anselmo Reggiardo
National University of General San Martín
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!
27th Jul, 2012
Singiri Reddy
Celon Laboratories Ltd
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.
27th Jul, 2012
Michael Mühle
ProBioGen AG
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 !
26th Jun, 2015
Bikram Pandey
AIT Austrian Institute of Technology
Thrombin is inefficient. I tried a lot to optimize the elution conditions. It did not work. Easier option is to clone into the vector with precission protease cutting site. This is way more efficient like Mathew said.
8th Oct, 2015
Syed Shahzad-Ul-Hussan
Lahore University of Management Sciences
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.
21st Mar, 2017
Ladislav Burysek
Protean s.r.o.

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