GST fused protein purification - (May/08/2007 )

Hi all,
I have a question about GST fused proteins.
Did anybody of you ever hear that putting GST to C-terminal of the protein gives trouble in purification step and is not efficient? Or you think this is completely not true?
A collegue told me this but could not explain why?
I would be happy to hear your ideas.
thanks

Hi Clementine,

I am currently trying to express and purify C-terminally GST-tagged proteins (~ 16kDa polypeptides). The trouble I have is a degradation product, that is GST-positive. Probably that is what your colleague meant, the free N-terminal could make it more sensitive to degradation. However, it is not impossible, they used C-terminal GST-tagged proteins in this paper and they produced quite a lot:
Mol Endocrinol. 2006 Aug;20(8):1838-52. Epub 2006 Mar 30

I am happy to share my experiences on purifying C-GST-tagged proteins, if you are interested, just please let me know.

There is no reason not to put a C terminal GST tag on your protein, provided that it will not disrupt its biological activity. The main reason GST is always an N terminal tag is because this is how all the vectors pGex, pET, etc are constructed. The vector pDEST24 is one of the only vectors I know of that has a C terminal GST tag but it uses the retarded gateway system. Of course you can easily make the primers to PCR the GST and place it in behind your gene of interest in pET or whatever. The purification should be every bit as efficient as putting it on the N terminus and no more sensitive to degradation. The cloning will just be not as straightforward.

Dear Krisztina,
Thank you very much for your email. I would be very happy to hear your experiences.
First of all, my proteins are about 110 and 90 kDa.
Would it be still possible to have GST on the C-terminal?
Thank you...
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Hi,

Although my protein is much smaller than yours, I don't see any reason why it would not be possible to C-terminally tag your protein. I don't think it makes a difference how big the protein is.
I will send you my protocol once I had it working properly. However, you may need to optimize it for your protein since a lot of others things can be far more important to consider when expressing a protein.