Your GST-fused His-Tag can be separated from the other two proteins by running your sample down a GST column (it should stick and your other two proteins should be in the flow trough). If the remaining two proteins have significantly different sizes, you can try to separate them by size exclusion chromatography (gel filtration). You can also try size exclusion chromatography without running the GST column first if all three proteins have very different sizes (or if at least your protein of interest is a different size from the other two). Also, if they have very different isoelectric points (pI), you can try separating them by ion exchange chromatography.

Hi there,

Glutathion agarose should retain the GST. Enterokinase might be removed using commercial kits like the following:

https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/prkebul.pdf

http://wolfson.huji.ac.il/purification/PDF/Protease_fusion_cleavage/Novagen_Enterokinase.pdf

And finally the protein of interest may be further purified on NiNTA.

Thanks Antonio Ariza and Dominique Liger,

@Antonio Ariza : The theoretical MW of porcine enterokinase (EK)  is ~22kDa. My protein is ~20kDa. So i was in doubt to go with size exclusion chromatography.

@Dominique Liger : My enterokinase is porcine while the kits were specific for bovine. Does this affect the removal? or can i proceed?

Secondly, Since i have to run series of different columns/purification steps, including the purification from host cell protein and desalting for purification. Does it affect my protein? (because many of the cases its just only NiNTA is enough to purify the protein. In my case there are several)

I don't know about crossreactivity of antibodies raised against bovine EK with porcine EK.... I don't know if it has been tested already. If not only the experiment would tell.