I don't understand: if you cut your fusion protein on the column, the GST moiety should stay on the column and you should recover your protein of interest only. Exclusion chromatography is not very resolutive, so I would not necessarily choose that purification step as a first trial, however: 1- it does not separate according to protein MW but according to Stokes radius which depends on MW and protein shape, so it could work with a very asymetrical protein; 2-GST is a dimer and roughly globular so it should elute as a globular protein of 52 kDa, and you might get some purification with a superdex 75 resin, for instance. In short, you should: check why you are recovering GST along with your protein (insufficient wash step, too harsh conditions?), then if you cannot prevent it, try other purification steps like ion exchange, hydrophobic or size-exclusion  chromatography.

Olivier

Plz see the attached file. Hope it will help

Thanks Sumit......

Maybe you can try molecular exclusion chromatography using a long high resolution column 

Thanks Gisele.....ya I will try that

You can remove the cleaved GST by another GSH-column step.

If I understand correctly, you have a GST-tagged protein linked with e.g. a 3C (Prescission) protease cleavage site. In that case you have two ways to proceed:

OR

I doubt gel filtration will separate 26 and 33 kDa very well, even with a long Superdex 75. Other than using an affinity column, your best bet is probably to use some form of ion exchange, especially if GST and you protein have very different pI.

I don't understand: if you cut your fusion protein on the column, the GST moiety should stay on the column and you should recover your protein of interest only. Exclusion chromatography is not very resolutive, so I would not necessarily choose that purification step as a first trial, however: 1- it does not separate according to protein MW but according to Stokes radius which depends on MW and protein shape, so it could work with a very asymetrical protein; 2-GST is a dimer and roughly globular so it should elute as a globular protein of 52 kDa, and you might get some purification with a superdex 75 resin, for instance. In short, you should: check why you are recovering GST along with your protein (insufficient wash step, too harsh conditions?), then if you cannot prevent it, try other purification steps like ion exchange, hydrophobic or size-exclusion  chromatography.

Olivier

Load your sample onto a HiLoad Superdex75 column, and set the flow rate at 0.6 ~ 1.0 ml.

Hi

Few months ago we had a similar problem. We used the following strategy:

1-we passed our GST-Protein through a PD10 column to eliminate GSH (from the first GSH-Sepharose column)

2- we loaded our GST-Protein onto protease-immobilized column

3- the resultant eluate (Protein, GST and non-cleaved GST-Protein) was loaded onto a second GSH-Sepharose column. For this step, we used a column with 1.5x the resin volume of the column used in the first affinity step (when we used a column with exactly the same volume, GST was not completely removed from the protein of interest)

It worked fine for us.

Emir

You can remove the glutathione bound by dialyzing against a buffer with a lower ionic strength than your buffer (if this is possible for your protein). After dyalisis you can perform a second purification step on the GST-column and your protein will be in the flow through of the column (and maybe an additional washing step to recover everything).

You can also perform size exclusion as GST normally elutes as a dimer (around 50 KDa).

By my understanding you cut the GST tag of your protein on the column and eluted the protein using glutathione.  I  did not understand why did you elute your protein after cutting it with protease because once you cut your protein of the GST-tag it should come in the flow-through which means the GST would be bound to the column itself. You need not elute it using glutathione instead just wash the column with 3 column volumes of buffer and your protein on interest without the tag will be in the flow through.

In brief the protocol should be: Apply your crude lysate with the GST-Protein on the pre equilibrated glutathione agarose column. Wash throughly with the buffer to remove any unbound protein. Now carry out the protease digestion on the column and collect the flow through. The flow through should contain you protein of interest without the tag.

I have done several GST-tagged protein purifications. For removing the GST-tag, I did this step on the column after the washing step, and before the elution step. After washing, I will put my protease in the equalibration and incubate with resin together for several hours ( according protocol ), and then I will collect the flow through which will contain my un-tagged target protein. Hope you good luck. 

Hi

I am doing same kind of experiment. 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?

Hi Rohini,

If i understand correctly you bound your purified protein to GST sepharose4B beads and carried out a on column cleavage. Its not always necessary that your on-column cleavage works because sometime binding of the protein to the beads may hinder the the accessibility of thrombin to its cleavage site due to which it may not cut. So to avoid this you can cut the protein in liquid before binding to the column and allow the cut protein to flow through the beads. By this the tag will bind to the bead and the cut protein without the tag will flow through.  I have been using thrombin cleavage very frequently and many time i have seen that thrombin does not work i.e its not active. So if by chance the thrombin that you used was inactive then the protein would not have been cleaved off the tag due to which the protein with the tag would bind to the column thus preventing it to come in the elute unless you elute it using reduced glutathione. Did the lot of thrombin that you are using work properly before? I use Human alpha Thrombin from Heamatologic Tech.

All the best

Anish

Hi,

I think the thrombin cleavage is quite slow comparing  with other protease like trypsin etc. Some time I have to do it overnight to get my protein-tag fully cleaved. You could also check the cleavage status with different enzyme ratio and different time.Then you know what is optimize conditions for the cleavage. If you want to do it off the column, after cleavage, you have to dialysis your sample against your GST equalibrious buffer, and then reload the sample to your GST-column., collect the flow through which contain your target protein.  

Hope it help

Weiping

Hi Anish

Thanks for the answer. I incubated by fusion rote in with GST sepahrose beads in a 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.

Sorry I meant fusion protein in first sentence

Hi Weiping,

Thanks for your reply. I incubated my fusion protein at RT for 1 hour, 2 hour, 4 hr and then 24 hours. I collected aliquots (protein in cleavage buffer+ thrombin agarose resin) and pass everything on column and simply collect elute. I see nothing in my elutes.

Hi Rohini,

Sorry about that. I am wondering do you have enough protein to work with? What I did was:

First I set up the test experiment. Usually I would set up 2 test tubes with 2 different ratios (one is 1unit thrombin to 100ug protein, and another one is 1unit enzyme to 50ug protein). I took the same amount of samples out from these 2 tubes at 0, 2h, 4h and overnight period, and once I took it out, just add the SDS sample buffer boiled for 5min. After all these conditions were done, I would have 8 samples plus molecular marker to run one SDS gel. Then I knew what my best conditions for this batch was.

Second I digested my real sample with thrombin following the conditions which I did on test run. I usually use at least 1mg protein to work with.

Thirdly, because the GST-resin have quite large capacity (around 7-10 mg protein/ml?), so you might only need small amount of resin, otherwise your sample might be too diluted. Also you could use Amicon (a centrifugal Filter Unit) to concentrate your sample. Sometime after concentration your protein bands will show up on the SDS-page. Hope these points help.

Wish you good luck

Weiping

Hi Weiping

Thanks for your help. Sure, I am repeating thrombin cleavage today as per your suggestions. Lets see...I hope to see something on my SDS PAGE.

Hi Rohini,

You are very welcome. Wish you good luck.

Weiping

Thanks to All......

I tried all ur suggestions with my protein and I getting my cut protein now....I have to improve little more...because I am still not getting complete cut protein its coming with uncut...but thanks to all....

thanks to Olivier and Anish.....sir I want to tell u that my cut protein is coming with uncut protein whn I am using glutathione...ideally the cut protein it should come in the flow-through....