Re: [ccp4bb] Off topic: 182 kDa protein is too large to crystallize

- Protein crystallography

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: Off topic: 182 kDa protein is too large to crystallize
From: Marek Frischerkase frischerkasemarek {- at -} YAHOO {- dot -} DE
Date: 2009-02-09

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Date: 2009-02-09

Subject: Re: Off topic: 182 kDa protein is too large to crystallize
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2009-02-09

Hi,

182kDa is large I'd say, but not too large for crystallisation.

Since you are using a Ni-column anyway you could re-clone it with a
His-tag that is cleavable by an enzyme (TeV, FactorX,... - I am a little
out of date about which enzyme is the most favoured on nowadays).

This would add one extra purification step, one with and an withouth
His-tag. Since these are complementary, this ought to yield already pretty
pure protein.

What buffer did you choose for the Gel filtration - did you have enough
salt to reduce interaction with the column? And what exactly do you mean
with 'interacts' - does the protein not elute at all? Same question holds
for the Ion exchange, what was the gradient you drove?

A "polishing" GF-step at the end of purification is definitely something
you want aim at.

Tim

--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

On Mon, 9 Feb 2009, Marek Frischerkase wrote:

> Dear all,
>
> I´m new in the field of crystallography. So please
> excuse my first question is off topic. I´m trying to purify a 182 kDa
> his-tagged bacterial enzyme. The expression in E. coli works quite
> well. After Ni-NTA the protein isn´t pure enough at all, so I tried
> several gelfiltration columns, but my enzyme interacts with the
> carbohydrate matrix, iex didn´t work either. So does anyone have
> suggestions? Perhaps a 182 kDa protein is too large to crystallize
> anyway?
>
> Cheers, Marek
>
>
>

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Off topic: 182 kDa protein is too large to crystallize
From: Marek Frischerkase frischerkasemarek {- at -} YAHOO {- dot -} DE
Date: 2009-02-09

Next message:
Subject: Getting Molprobity to run using WinCoot
From: ANDY DODDS andy {- dot -} dodds6 {- at -} GOOGLEMAIL {- dot -} COM
Date: 2009-02-09