Re: [ccp4bb] Zinc or Iron binding protein, that is a question!

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

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Subject: Re: PHASER packing function with DNA
From: LEDU Marie-Helene 161111 Marie-Helene {- dot -} LEDU {- at -} CEA {- dot -} FR
Date: 2009-08-06

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Subject: Re: Zinc or Iron binding protein, that is a question!
From: Marius Schmidt marius {- dot -} schmidt {- at -} PH {- dot -} TUM {- dot -} DE
Date: 2009-08-06

Subject: Re: Zinc or Iron binding protein, that is a question!
From: Xuan Yang pattisyang {- at -} GMAIL {- dot -} COM
Date: 2009-08-06

Dear Mr. Fritz,

Yes, the protein is not an E.coli protein! Instead, it was cloned from a
virus. And since it was a nonstructural viral protein, I thought it might be
appropiate to treat it as eukaryotic proteins.

E.coli system was quite different from eukaryotic ones, hence I was quite
cautious about the ICP-ES result and trying to confirm it via alternative
method. Thanks very much for mentioning the examples which suggested that Fe
might be contaminants. Indeed, when I cut the protein in two parts (still
with MBP) and test them via ICP-ES again, Fe became negligible in both and
Zn stoichiometry increaed to 1:1 in the C-terminal part. The result lead me
to focus on Zn instead of Fe. But I still want to confirm the idea.

Matallo biochemistry was exactly what I dreamed to do.

Sincerely,

Xuan Yang

2007/8/6 Guenter Fritz

> Hi Xuan,
> I guess your protein is not an E.coli protein. There are several examples
> that eukaryotic Zn-proteins expressed in E.coli contain Fe instead of Zn. I
> am sceptic whether IMAC with different metal ions will give the solution of
> the problem. If you really want to get information on the metal ion binding
> properties you will have to do some matallo biochemistry: preparing apo
> protein, reconstitution with metal ions, UV-Vis spectroscopy, EPR would be
> great, ...
>
> Dear Sir or Madam,
>> The ICP-ES results indicated that 1 molar my protein purified from E.coli
>> Origami(DE3) contained about a half molar Zinc and nearly a quarter molar
>> Iron (whether II or III was not available). The protein carried a MBP tag on
>> the N-terminal and the situation was similar with or without His tag at the
>> C terminal. I want to determine whether my protein really bind Zinc or Iron.
>> Does anyone have any experience about such problems?
>> Specifically, now I want to compare the binding efficiency on various
>> IMAC, i.e. 50mM ZnSO4, FeSO4, Fe2(SO4)3, NiSO4(control), or CuSO4(control).
>> However, considering the instability of Fe(II) in solution, the design
>> still seemed problematic.
>> Sincerely,
>> Xuan Yang
>> National Laboratory of Biomacromolecules and
>> Center for Infection and Immunity,
>> Institute of Biophysics,
>> Chinese Academy of Sciences,
>> Room 1617, 15 DaTun Road,Chaoyang District,
>> Beijing, China, 100101
>> Tel: 86-10-64884329
>> Academic email: YangX@moon.ibp.ac.cn
>> We will either find a way or make one.
>>
>>
>
>

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

Previous message:
Subject: Re: PHASER packing function with DNA
From: LEDU Marie-Helene 161111 Marie-Helene {- dot -} LEDU {- at -} CEA {- dot -} FR
Date: 2009-08-06

Next message:
Subject: Re: Zinc or Iron binding protein, that is a question!
From: Marius Schmidt marius {- dot -} schmidt {- at -} PH {- dot -} TUM {- dot -} DE
Date: 2009-08-06