GST protein purification - only 3% bind to beads (Dec/10/2005 )

Hi,

I got a headache at purifying GST fusion protein. The fusion protein (39kDa) was found in the inclusion body and solubilized with sarkosyl, the sarkosyl was subsequently quenched with 2% triton. But I found only less than 5% total protein was bound to glutathione beads, even with overnight incubation.

However, when I performed a parallel experiment with exactly same process but using induced GST protein, I found more than 70% could be captured on the beads.

Any suggestions? thank you so much!

hi
probably induced means salts and pH equilibrated.
maybe you may use an i trap desalting column before doing purification...

Hi Bullfrog,

Perhaps the GST part of your fusion protein hasn't refolded properly after solubilisation. Unlike His tags, GST tags need to be properly folded to bind to resins and magnetic beads.

Bill

hi bullfrog and everyone. i was searching the forum and found this thread....i have excatly the same problem.....please can you tell me how you have solved it? thanx a lot

.i have excatly the same problem

hi

using column purification for GST is better than beads

another way is to decrease the flow rate thru the column,, slow elution

then recirculate the lysate several times to increase the binding ( that worked for me once)

then u can also try increasing the concentration of glutathione elution buffer from 10 mM to maybe

15-- or 20 or even 50mM
also check the pH ,, keep it between 8-9


increase the elution ,, meaning do elution 5-7times ,,, or more if u want to


all this information can be found at website for amersham ( now GE )for GST columns
serach for GST fusion system handbook

i am attaching some good protocols i got from the site and some where else
hope its useful

Hi:
According to your said, i concluded that the GST fusion protein is partial correct refolding (about 5%), The solution is to increase the supernatant expression level by optimization of temperature (lower, i once lowed to 15 degree), concentration of IPTG (lower), induction time (lower) etc.
good luck!