Purification protein from inclusion bodies - (Nov/27/2006 )

Hi everybody!!

I have a problem with the purification of my protein and if anybody could helps me I would be very grateful.

I'm trying to purify a recombinant protein, which is expresed in E. Coli (Bl¡L21DE3) by Ni column, due to my protein goes to inclusion bodies I'm using buffers with SDS.

The problem is that I purified a protein with a bigger molecular weigth that I expected (I expected around 20 kDa and my protein is around 30 kDa) , and also my protein doesn't have inmunological activity, and I don't know if it is a problem relation with the folder of the protein or could be another thing.

I'm waititng for your advice.

Thanks a lot!!!

Ana.

---

A few questions/comments.
1. Is the codon usage optimised for your protein? Check it at http://gcua.schoedl.de/. If not, try a different strain, like the Rosetta or one of the other commercial variants, and you might find that your protein is soluble.
2. Try using sarkosyl, rather than SDS, and the proteins might solubilise easier.
3. Are you using urea to denature the inclusion body?

Good luck.

---

please use sarkosyl, if that doesnt work use urea and then you have to dialyse your protein (refold it) to get back its activity i guess. i dont think using SDS is a good idea.

---

Thans for your comments. I will do these changes and I hope these work.

Thanks.

Ana.

---

HI Kathy,

I have some question which is the different between sarkosyl and SDS, and why do you think that SDS is not a good idea, perhaps it's very strong? Because the problem is that I don't have sarkosyl in this moment in my lab and I need to purified my protein in an active way as soon as possible.

And I don't have any experience in dialysing of protein could you give me some protocols or some advices about that?

Thaks a lot.

Ana.

---