Thrombin cleaved GST-tagged protein remained bound to beads!
Posted 25 April 2011 - 05:06 PM
Recently I have had a hard time to purify my protein.
My fusion protein construct is inserted to PGEX2T vector which means my protein is GST tagged. The Mw for my protein is about 90kD so plus GST, it should be around 116 kD.
I transformed the plasmid to BL21 (DE3) [Also I transformed to Rosetta Gami series strain but no difference in expression]. I can clearly see the induced expression. One problem is most of my protein, is actually in inclusion bodies. But that's fine.
According to GE Healthcare purification handbook, I dissolved the pellet with cold 1x PBS, treated with protease inhibitor and sonicated the cells (15s pulse, 5min on ice, repeat for about 8-10 times). Then I added Triton X-100 to final conc. 1% and then centrifuged my sonicates at 10000rpm for 40min. Then I collected the supernatant and mix it with Glutathione Sepharose 4B (GE Healthcare) for 4 hour binding. Then I spin down the beads and collect the supernatant (this fraction, compared to the one before binding, should have less my target protein 116KD. BUT I rarely can tell from the gel. The concentrations are indeed different).
After several washes with PBS, I did an in situ cleavage with thrombin (which means directly in the column) at RT for about 16 hours. (I also did 4 hours and succeed to get some proteins out for some cell culture experiments).
Then I collect the eluates for 5 times and analyzed by BCA assay and SDS-PAGE.
Surprisingly, I found almost no my target protein (90KD) in every eluate. And almost all my protein remained bound to the beads.
I don't know why, probably the hydrophobic region of my protein interact with the sepharose beads.
I don't know if any of you have similar experience or someone has some sort of solutions, please let me know. Thank you.
I attached one of the images so that you can understand better. Thank you!
Lane 1: protein ladder
Lane 2: supernatant before binding
Lane 3: beads before thrombin cleavage
Lane 4: supernatant after binding
Lane 5 Lane 9: eluates after thrombin cleavage
Lane10: final beads after 5th wash
Posted 25 April 2011 - 10:22 PM
Edited by protolder, 25 April 2011 - 10:24 PM.
Posted 26 April 2011 - 06:47 AM
Hola I never has done it but some experts in this forum recommended digest the fusion protein outside the colum and after separate both fragments in the column. Check anterior messages here if you see something or use the forum searcher. Have you induced at low temperature about 25šC in order to have more solule protein? Buena suerte
Thank you. I induced the protein expression at 23 degrees for 3 hours.
Yes, I will try to elute the GST fusion protein out and do thrombin cleavage in solution to if I can have better results.
One possibility that the hydrophobic interaction between my target protein and the beads happened at the first time when I added the beads, then I would not be able to elute out with reduced glutathione...
Posted 27 April 2011 - 09:35 PM
Posted 28 April 2011 - 08:13 PM
Hola, you could modify the buffer untill 0.5-1M NaClfor polar interactions and adding 10% glycerol for hydrophobic forces, but firstly check the compatibilites of colum that now I donīt know. Buena suerte
Thank you so much.
Yesterday I eluted the GST portion out with elution buffer (10mM reduced GSH in 50mM Tris HCl) and today I used 1M NaCl trying to wash the protein out and I will keep you updated tomorrow.
I don't get it when you say adding 10% glycerol for hydrophobic forces. Where to add glycerol?
Also you said checking the compatibilities of the column, would you please elaborate a little bit?
I appreciate your help.
Posted 28 April 2011 - 09:52 PM
Posted 29 April 2011 - 07:49 AM
hola, I have checked the gelifesciences to see the characteristic of glut.sepharose 4B, in the part of chemical stability, it says that there isnīt appreciable lost of activity with the exposure for short time to 0,1M NaOH, 6M HCL guanidine or 70% ethanol, nothig about glycerol, but I said to add these to the buffers in order to increase hydrophobycity . I hope donīt confuse you but with other tagged proteins such components are added to the buffers to improve elution, I have use for instance 10% ethanol or glycerol in His-tagged proteins with good results. if this ressin suports 70% ethanol if the increase of salt doesnīt run ok, 10% ethanol in the buffers coud be an alternative but you have to do the try, have you any no packed reson to do a little in batch try? one more important thing in the recommendations the manual says add 1-20mM DTT to avoid oxidation of GST wich drives to the aggregation of fusion protein .could be this the problem? if your in course experiments if your protein continues inside the colum make a wash with your buffer with 10mM DTT and see if any part of your protein elute, if yes continue washing untill elute all. Have you the manual? check these indications or check http://www.gelifesciences.com/aptrix/upp00919.nsf/Content/678E31D37FA9E19DC1257628001CCE08/$file/18114275AD.pdf Buena suerte
Thank you soooo much.
Let me share some more images with you.
So I had two 500 ml cultures, one of which I used GST buffer which contains the ionic detergent I mentioned above to disrupt the inclusion bodies and the other I just used PBS to do the job.
As you can see from the first two images, there are binding and cleavage but most of the protein of interested were in the last lane which is the beads after 5 washes.
Then I decided to use elution buffer (50mM tris cl and 10mM reduced GSH) to elute GST out and you do see a lot of GST coming out (26kD). The first 4 lanes after the ladder are from the one I used with GST buffer and the latter 4 lanes is from PBS extraction. It looks like in the PBS extraction there is still my protein bound to the beads but I barely see that in the GST buffer extracted one.
Then I was thinking since the GST portion has come out, I can salt out my protein. So I eluted the two columns with 100mM NaCl twice and 1M NaCl for the third time. However, it looks like there is no protein of interest in the beads in the 5th lane which is the one I extracted with GST buffer but there is still protein bound to the beads I extracted with PBS. But neither of these show protein of interest on the gel.
I only load 20ul protein so the conc. may be too low that I cannot detect with this method. But I don't know the exact reason. Maybe it didn't go out with NaCl.
So next time I will try your suggestions.
Also I am wondering whether I should buy B-PER solutions from Pierce which is designed for inclusion body proteins.
For the ethanol, glycerol or DTT, are you saying I should add to PBS?
All in all, thank you so much.
Posted 29 April 2011 - 10:13 AM
genius does what it must
i do what i get paid to do