GST fusion protein purification - (Jul/11/2006 )
Hi, All:
The size of my GST fusion protein is around 80KD(including GST tag). After purification from column, I always see the cleaved fusion proteins from the eluted fractions, even i added protease inhibitor cocktail and PMSF during lysis the bacteria pellets. So, how do i avoid this problem?
Sometimes your construct is partially degraded during the induction step within the bacteria, it just happens for some constructs; so you purify all the GST tagged products (complete and incomplete). You should check in your raw lysate the integrity of your fussion protein. It can be difficult to see it in a Coomasie staining. Have you tried an anti-GST blott of your raw lysate?
Miguelon
hi,
miquelon is right, first perform western blot of GST in both lysates and purifies portions of ur protein.
then u will be in a position to decide where ur protein is getting cleaved then u can find out the solution for it.
gud luk
payeli
The size of my GST fusion protein is around 80KD(including GST tag). After purification from column, I always see the cleaved fusion proteins from the eluted fractions, even i added protease inhibitor cocktail and PMSF during lysis the bacteria pellets. So, how do i avoid this problem?
yes, i did run coomasie and western blot to check the expression of gst fusion protein from whole cell lysate. It's very hard to tell there's cleavage occured by coomasie stain. But by western blot, i saw quite amount cleavage from my fusion protein. Becuase I am going to use the fusion protein to pull down other interacting proteins, i just worry about the sensitivity issue. Any way, since i also have another tage at the c-terminal of this fusion protein, do ur guys suggest me that i could connect with another column to decrease the amount of cleaved protein in the fraction?
I think it´s a good idea. What kind of tag is it? The only thing is that you should take care not to use denaturing conditions if you planned to rebind your fussion construct to glutathione beads, because denatured GST cannot bind and refolding is not 100% efficient?
Maybe you could use the second affinity matrix for the pulldown?
Miguelon
You could also try to improve your expression conditions in a few miniscale preps, to minimize that cleaving. I think there are some recent posts on the subject. In my case, it helped me a lot to express my fussion construct at low temperature O/N. Good luck
Maybe you could use the second affinity matrix for the pulldown?
Miguelon
yes, i did run coomasie and western blot to check the expression of gst fusion protein from whole cell lysate. It's very hard to tell there's cleavage occured by coomasie stain. But by western blot, i saw quite amount cleavage from my fusion protein. Becuase I am going to use the fusion protein to pull down other interacting proteins, i just worry about the sensitivity issue. Any way, since i also have another tage at the c-terminal of this fusion protein, do ur guys suggest me that i could connect with another column to decrease the amount of cleaved protein in the fraction?
thanks lot, anyway, could u tell me what sort of temperatures i should start with if i want to use for expression my protein O/N
It worked quite good for me to induce for 15 minutes at 37º at optimal OD with high amounts of IPTG (1mM) and incubating O/N at 18ºC. I´ve seen protocols that even go lower (16ºC), and somebody in the forum said she had seen protocols that incubate at room temperature. Anyway, check past posts of the forum because there where lots of opinions about the subject, not only temperature, and there were suggested some very good websites as well.
Good luck.
Hi, Maybe you can use C-terminal GST tag. I don't know any but htere should be some!!
use nickel column kit. Promega