[Protein-analysis] RE: zinc finger protein purification

David Bienvenue via proteins%40net.bio.net (by DBienvenue from vlstcorp.com)
Fri Jan 15 12:41:18 EST 2010


Zongwen,

What buffer system are you concentrating the protein in?  What is the isoelectric point(pI) of the protein, compared to the pH of your buffer (generally proteins will be less stable at pH's near their pI).  I would recommend a change in buffer- we have had good success staying away from PBS, and using 10 mM Histidine, pH 6.5, with additives to help stabilize the protein.  Excipients like Polysorbate-80 (Tween-80) in low concentrations (0.03% w/v), sucrose and mannitol are all helpful in minimizing aggregation at higher concentrations.

Good luck!

David Bienvenue, Ph.D.
Senior Principal Scientist
VLST Corporation

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Sent: Friday, January 15, 2010 9:05 AM
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Subject: Proteins Digest, Vol 56, Issue 5

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Today's Topics:

   1. zinc finger protein purification issue (???)


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Message: 1
Date: Fri, 15 Jan 2010 15:33:25 +0900
From: ??? <jin.zongwen from gmail.com>
Subject: [Protein-analysis] zinc finger protein purification issue
To: proteins from magpie.bio.indiana.edu
Message-ID:
	<4153bf441001142233u182fd76bu92773772fda0c2ca from mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1

I recently started to purify a protein containing zinc finger modif.

the protein consist of flexible linker and his tag in the N term and C
terminal of the mofit.

the Zinc finger itself is consisted from three zinc finger motifs.

It expressed well in the E.coli, and I was able to purify the protein, but
when I tried to concentrate it by using a membrane filter( amicon centricon)
the protein start to aggregate.

what is more a concern is that gentle swiring in 4'C would generate cotton
like aggregates in the tube.

I would like to ask if anyone have expperience on zinc finger protein,
expecially small zinc finger proteins like this one.(15kDa)

any suggestion are welcomed.

Best regards to every one

-- 
Zongwen Jin
Biomolecular Engineering Lab
Dept. of Biological Sciences, KAIST
335 Gwahangno, Guseng-dong, 305-701
Yuseong-gu, Daejeon, Republic of Korea
Researcher ID: B-4590-2009
Lab: +82-42-350-2656
Mobile: +82-10-7460-9518
E-mail: sutem from kaist.ac.kr
K-Internet : 070-8288--0606


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