GST Tag Cleavage - (Nov/29/2005 )

Hello all,

I recently expressed a GST-Tagged protein and purified it with gluthathione sepharose. I then added trypsin to the beads in order to cleave the protein from the GST-tag and recover just the protein, but I do not know how to separate the trypsin from my protein of interest. Please help!!!

-Ken

the trypsin is a much lower concentration than your target protein(you won't even see it on a gel)and therefore should not pose a problem for downstream applications. if you need a really pure sample then purify your protein further with an anion exchange or size exclusion column or even possibly dialysis.

hope that helps!

hi,

ion exchange chromatography is a pretty easy way to accomplish what you're looking for, that is, unless your protein has a similar pI to trypsin. hydrophobic interaction chromatography might work too...again, it depends on your protein. also, you might want to look at some catalogs and see if they sell some sort of His tagged or GST-tagged trypsin. if so, you can make use of that by running your cleaved protein/trypsin mixture over Nickel or glutathione resins and collect your protein of interest in the flow-through, while trypsin remains bound to the resin.