Purification of GST-fusion - GST-fusion protein: How to express and purify (Feb/09/2007 )

I have taken ~30 amino acids of a protein and fused it to GST in pGEX 2T and want to use it as a kinase substrate. Does anyone know how to express this thing in bacteria (they are already in DH5 alpha cells) and purify it?

Thanks
DOCDTT

-DOCDTT-

Use Ni-Sepharose column for purification.
Use thrombin enzyme to cleave off your GST.

Check out Amersham Bioscience for more details about GST system.
You can get free download from here.

http://www1.amershambiosciences.com/aptrix...News~GSTHandb02

-timjim-

I hate to be pedantic, but you'd be better served by using a Glutathione (GSH)-Sepharose, rather than Ni. Ni is for His tag systems, not GST tag

-swanny-

And you'd be better off using BL21s than DH5s.

-jaknight-

QUOTE (jaknight @ Feb 11 2007, 05:46 AM)

And you'd be better off using BL21s than DH5s.

Hi all,
Thanks for the replies.

I'll be certain to use GSH-sepharose - I hope I know that much. As for the cells, I guess it's BL21. The post-doc in my lab says DH5 - is she absolutely wrong or are BL21's just better? If she is, then I am having major doubts about her...

DOCDTT

-DOCDTT-

BL21 is an expression strain. It gives better protein yields but could mess up your plasmid. DH5 is a cloning strain. Plasmid integerity is better maintained but protein yeild would probably be less.

It not absolutely wrong. One could use DH5 cells but BL21 would probably serve you better

-perneseblue-

QUOTE (perneseblue @ Feb 11 2007, 02:02 PM)

That's the ticket...Thanks perneseblue

-DOCDTT-

BL21 has some ohm- gene (protease encoding gene) i think. That's why it will be better choice for expression. Am I right? Just want to make sure.

-timjim-