4 replies to this topic

[mervyn]

hi all,

Recently  I met a problem in purification of GST fusion protein,  the protein is expression and soluble, but can not bind the GST column, all in the flow through. I use the PBS(pH7.4) as the binding buffer, and elution buffer is GSSH(pH8.0). I already check purified ability of this column by run protein from pGEX-4T1 empty vector, and everything is ok.

And then I tried the his-tag construct, also got the soluble protein , but can not bind the HIS column.

The PI of my protein is 9.5, so if should I change the binding buffer's pH?

Everybody, Please recommend to me how to solve this problem.

Thank you very much

mervyn

[mdfenko]

is the gst exposed? is it c-terminal or n-terminal?

the pI of the protein should have no effect on the binding of the tag.

[mervyn]

mdfenko, on Nov 7 2009, 12:45 AM, said:

is the gst exposed? is it c-terminal or n-terminal?

the pI of the protein should have no effect on the binding of the tag.

My protein is ~43KD, the Gst is in  N-terminal, and how can I check the gst exposed or not?

Thanks

[mdfenko]

you would have to know something about the 3d structure of the protein.

but, you could make a new fusion protein with c-terminal gst and see if that binds.

or you could denature your protein with urea, run it on the column then renature (you may get a reduced yield this way).

[mervyn]

mdfenko, on Nov 10 2009, 01:03 AM, said:

you would have to know something about the 3d structure of the protein.

but, you could make a new fusion protein with c-terminal gst and see if that binds.

or you could denature your protein with urea, run it on the column then renature (you may get a reduced yield this way).

I have changed the binding buffer's pH, but also got poor binding.

I will do the crystallization  in the next step, so the renaturation is not suitable.

And I want do a new construct with N-terminal gst tag and C-terminal his tag, and use HIS colunm for purification.

Thanks.

mervyn