Protein purification from greenish supernatant of Pichia pastoris - (Jun/28/2013 )

Hi,
I'm working with Pichia. I'm trying to express diferent proteins, and in each case I have the same problen in the purification fase.
After fermentation the supernatant is greenish as I know it's normal, because AOXI forms crystalloids with FAD, which is producing in the supernatant by cell leakage in the bioprocess.
The problem is that when I try to separate this crystalloids from my protein by TFF or gel filtration I can't do it because my protein is agglomerated with this complex, and I can't find the way to disaggregate it.
Anyone can help me with this?
Thanks!

what have you tried to disaggregate the complex?

non-ionic detergent? reducing agent (bme, dtt)? ethylene glycol or peg? urea (then purify and renature)? a combination?

mdfenko on Mon Jul 1 16:03:01 2013 said:

Hi mdfenko,
I've tried with non-ionic and ionic detergent (tween 20 2% after fermentation and during fermentation 0,05%) ( SDS 0,15% after fermentation). Urea 5M, after that, I use TFF to separate and then analyze by Dot-blot or Western-b. In no case could separate the agglomerates.
The only evidence I have of disaggregation is by anionic exchange, I see a part of agglomerated protein is separeted of the agglomerate, is it possible that the interaction with the matrix of exchange produce the disaggregation?
I must find the cheapest technic because the product is to veterinary use.

i assume you used a salt gradient or step to elute the protein, maybe salt separated them?

or did you change pH to elute? if so, then maybe you can separate them by changing pH.

mdfenko on Wed Jul 3 12:12:00 2013 said:

Yes, I've used salt step to elute the protein. But I want to solve the problems of the aggregates before chromatography. Do you have any idea with this data to solve that? Thanks!

if salt is what broke up the aggregate then add salt to the protein solution before chromatography. then you can separate the protein by gel filtration in the high salt medium.

mdfenko on Wed Jul 3 15:12:10 2013 said:

Yes I've tried that but the aggregate still equal. The disaggregation only it's seen when is used the anionic exchange matrix.

then it would seem that you will have to clean your protein with anion exchange. sorry.

Is there any particular reason you need to express your protein in Pichia?

If you are purifying native protein, this leads me to a second thought. The fact that denaturing detergents, non-denaturing detergents, denaturing agents, reducing agents, high salt concentrations and pH adjustments cannot effectively separate your protein from the agglomerates may be telling you something about your protein. These agglomerates may be required for stability or function of your protein. In which case, do you have any reason to believe you should be able to separate these pieces?

labtastic on Sun Jul 7 22:58:58 2013 said:

Thanks for the comments, we need express in Pichia for the low cost.
We know that the agglomerate contains proteins of pichia and the recombinant protein. The main evidence that the pichia's proteins are responsable of agglomerate is that it's occurs with different recombinant proteins.