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Protein purification from greenish supernatant of Pichia pastoris

Pichia pastoris greenish

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10 replies to this topic

#1 recuperomn

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Posted 28 June 2013 - 09:04 AM

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!

#2 mdfenko

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Posted 01 July 2013 - 08:03 AM

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?
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#3 recuperomn

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Posted 02 July 2013 - 06:34 AM

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?

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.

#4 mdfenko

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Posted 03 July 2013 - 04:12 AM

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.

Edited by mdfenko, 03 July 2013 - 04:12 AM.

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#5 recuperomn

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Posted 03 July 2013 - 07:04 AM

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.

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!

#6 mdfenko

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Posted 03 July 2013 - 07:12 AM

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.
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#7 recuperomn

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Posted 03 July 2013 - 07:33 AM

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.

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

#8 mdfenko

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Posted 04 July 2013 - 08:46 AM

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

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Posted 07 July 2013 - 02:58 PM

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?

#10 recuperomn

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Posted 07 July 2013 - 05:24 PM

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?

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.

#11 labtastic

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Posted 18 July 2013 - 07:05 AM

You say you need to express in Pichia for the low cost...I'm not sure what the alternatives are, but it would seem to me your boss if paying you quite a bit money for the time it has taken you to trouble shoot this problem without much success and poor yield. Sometimes investing a little extra $$ upfront will save lots of time and $$'s down the road.

I had a boss in grad school that insisted I spend weeks developing an assay or protocol with home-made materials so that he wouldn't have to spend the $200 for the kit. So instead of paying $200 and having the results in 2 days, he paid me for four weeks of labor and time before getting any resuls. Basically, my boss ended up losing both time and money in the whole process, thinking he just saved a bundle by not buying the kit. This is what I would pitch to your boss if this were my project. :)




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