GST purification - general protocol (Aug/09/2005 )
Hi all, I'm a chemist and I've been using some GST-purified enzymes (made by someone else) as a reagent in the lab, and I was wondering if anyone could give me a rundown of what is involved in the steps before they get to me!!!
it depence what kind of protein it is....Are you interested in the steps during protein production or what? Also the purification conditions may be different...The best is, to contact that lab. But are some commonly used steps used at work with proteins, but it can be variable...and, I can also send you the Amersham manual (working with GST tag),if you want.
Good luck, Ell
Since I'm waiting for my GST-fusion protein to filter through my columns, let's give you a quick rundown (if you haven't just read the pGEX manual from Amersham, that is.)
1. Clone the insert into a suitable vector containing an multiple cloning site directly after the GST coding sequence. Make sure the insert is in the right frame!
2. Transform the expression vector into BL21 (DE3) or another suitable E.coli expression strain, or into another expression system if that is your fancy
3. Grow the bacteria until an appropriate population number (OD=0.6 is good) and then induce the expression of the fusion protein. Induce for the applicable number of hours, this is temperature dependant.
4. Harvest cells, resuspend in a small volume of fex. PBS, and lyse the cells by sonication or freeze/thaw. Sentrifuge the lysate hard! Now is a very good time to find out if your protein is soluble (in the supernatant) or insoluble (in the pellet). For convenience the protein should be soluble.
5. Purify the protein from the lysate supernatant. If done by columns, simply apply some ml's of glutathione sepharose to a column, wash 3 times with PBS and the apply the sample. Then wash again with PBS, before you elute with reduced glutathione. The elution should go over at least 10 minutes, possibly just put the caps back on the columns and let the columns with glutathione stand overnight at 4 degrees.
6. Collect the eluate. Dialyse to get rid of the glutathione, perform buffer exchange if needed.
7. Deliver the purified GST fusion protein to the next downstream user.
And that's it!