Cleaving a protein from GST - Troubleshooting methods to liberate my protein from gluthathonine sepharose (Aug/08/2006 )

Hello all,

I am having a problem in cleaving my disaster of a protein from GST using both Thrombin and Precission Proteases. Ok, maybe the problem isn't necessarily the cleavage but liberating the "cleaved" protein from the glutathonine sepharose. I have tried various elutions methods like using various concentrations of NaCl and Triton-X; 1M and 1% being the highest concentration used, respectively. Also, I've used various elution times with 24 hours being the longest. As a final straw, we have even used 1% SDS and boiling the sepharose to liberate my cleaved protein. After many comassie and silver stained gels later, it appears that the cleavage works because there's a correctly sized band for my protein; but most of the protein remains in the sepharose (I would say that I elute at the most about 5-10% of my protein). I have even tried to do the cleavage step on the bacterial lysate prior to incubation with the glutathione, with the same results. Oh the lab uses the batch purification glutathonine sepharose from Amersham, GE life, or whatever the hell they go by nowadays. Would it be better if I used the column system, which my PI thinks is worse?

Please help, my antibody development and ultimately my PhD depends on this

Thanks

so you are assuming there is some sort of interaction between your protein and the sepharose.
have you tried a different carrier for the gst, GST beads for example, might relieve the problem



do you need a functional protein for your antibody?
could try urea
could try altering pH and tmeperature protein this may weaken any possible interactions, though increasing temperature might also kill your protein!!


just a couple of ideas

enjoy

Hi swosei12,
If you have previously expressed the protein but ended up in an inclusion bodies, then I will recomend this and it worked excellent for my antibody production:
1. Use 1%CHAPS to ws your cell pellet several times until the inclusion bodies are clean from many proteins.
2. Run a Coomassie gel to check for purity on the cleaned proteins after solubelizing with detergent.
If its clean enough, 70-90% protein enriched, then run a preperative SDS gel electrophoresis and partially stain the gel.
3. Cut the band of interest and "Electroelute" using a MWCO size filter.
4. Concentrate the debatured protein and rerun it again on a similar preparative gel electrophoresis. Do a longer run.
5. Repeat steps 3-4 again.
6. COncentrate and estimate the amount by comparing with BSA on gel. Your protein will still be blue in color.. thats OK, simply immunize the rabbits with this protein.
I tried it twice with two different proteins from Inclusion bodies and the antibody I got was excellent.
Good luck
Ameer

I've done several GST protein preps and it is a common practice in the lab which I am currently working with. Protein yield due to cleavage problems has been a off and on problem. One thing that we discovered is that thrombin needs CaCl2 (10mM) in order to cleave efficiently. We do all of our binding and washing in a 50 ml conicle tube and then we actually cleave the protein by loading our beads into a collumn and adding thrombin (in a specific buffer) and incubating for 4 hours before collection. I can give you the protocol if you are interested.