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Gst tag purification problem - (May/01/2006 )

Recently I am working on a human protein, the size is about 50kD. This protein is absolutely unsoluable with the his tag in the pET competent cells. So I tried to add a Gst tag on it to try to improve its solubility with the pGEX vector. It seems solved in the cell free and I could test some activity with it. However the problem is there is a very big Gst (26kD) band in the cell free. So after I loaded on to the Glutathionine 4B sepharose column, most of the portein bound on it is only the Gst wothout my protein. I have sequenced the gene construct, it is completely right with the Gst on it. So is there anybody met the the same problem? How could I avoid it? Thank you very much!

-ruiwu-

if your gst-protein is not binding because of the excess of gst alone then you could recycle the wash through the column and have your protein bind after depletion of the gst alone.

-mdfenko-

hello,

sometimes a protein might be soluble in whole cell extracts, but will precipitate when concentrated on a column. that can be a pain. detergents might help you there (anything but SDS).

also, if you have some degree of vector contamination, you might be co-expressing free GST with your GST fusion protein. if that's the case, you can try to go back to your cloning steps and make sure you're only tranforming with purified vector that contains your insert.

another thing to try is a quick separation of your fusion protein from free GST through ammonium sulfate precipitation. it's likely that they'll have different solubilities, given your protein's poor solubility. you could grow up a 5ml culture, lyse it, collect your soluble extract and do the A.S. cuts in a day and have your answer. if the two proteins appear in different pellets, use the one that has your protein of interest. if this doesn't work, maybe try something else, like anion exchange chromatography.

once you've separated the two, then perform your glutathione affinity purification step.

still, i would look at your vector stocks first before setting out on a multi-step protein purification.

i hope this helps.

if you have more questions, feel free to email me at jonmike.reed@gmail.com

good luck.



QUOTE (ruiwu @ May 1 2006, 06:06 PM)
Recently I am working on a human protein, the size is about 50kD. This protein is absolutely unsoluable with the his tag in the pET competent cells. So I tried to add a Gst tag on it to try to improve its solubility with the pGEX vector. It seems solved in the cell free and I could test some activity with it. However the problem is there is a very big Gst (26kD) band in the cell free. So after I loaded on to the Glutathionine 4B sepharose column, most of the portein bound on it is only the Gst wothout my protein. I have sequenced the gene construct, it is completely right with the Gst on it. So is there anybody met the the same problem? How could I avoid it? Thank you very much!

-johanski-

QUOTE (johanski @ May 3 2006, 09:28 AM)
hello,

sometimes a protein might be soluble in whole cell extracts, but will precipitate when concentrated on a column. that can be a pain. detergents might help you there (anything but SDS).

also, if you have some degree of vector contamination, you might be co-expressing free GST with your GST fusion protein. if that's the case, you can try to go back to your cloning steps and make sure you're only tranforming with purified vector that contains your insert.

another thing to try is a quick separation of your fusion protein from free GST through ammonium sulfate precipitation. it's likely that they'll have different solubilities, given your protein's poor solubility. you could grow up a 5ml culture, lyse it, collect your soluble extract and do the A.S. cuts in a day and have your answer. if the two proteins appear in different pellets, use the one that has your protein of interest. if this doesn't work, maybe try something else, like anion exchange chromatography.

once you've separated the two, then perform your glutathione affinity purification step.

still, i would look at your vector stocks first before setting out on a multi-step protein purification.

i hope this helps.

if you have more questions, feel free to email me at jonmike.reed@gmail.com

good luck.



QUOTE (ruiwu @ May 1 2006, 06:06 PM)

Recently I am working on a human protein, the size is about 50kD. This protein is absolutely unsoluable with the his tag in the pET competent cells. So I tried to add a Gst tag on it to try to improve its solubility with the pGEX vector. It seems solved in the cell free and I could test some activity with it. However the problem is there is a very big Gst (26kD) band in the cell free. So after I loaded on to the Glutathionine 4B sepharose column, most of the portein bound on it is only the Gst wothout my protein. I have sequenced the gene construct, it is completely right with the Gst on it. So is there anybody met the the same problem? How could I avoid it? Thank you very much!

-susanrui-

Thanks for everybody's kind suggestions. This protein really made me crazy, I 'll check the vector stock first. Thanks.

-susanrui-

Hello!
If I understand, you have a construct of your protein with GST, and you express it in the right size, but after binding to the Glutathione resin you elute mainly GST alone?
I had similar problems with some proteins ( I produce a correct size construct but after elution I have only GST) that I am working with and unfortunately I have not found the solution, but I think that I have problems of degradation after the lysis of the cells. Maybe adding an inhibitor cocktail (I use on from sigma) will help you
Good luck

-rumo-