Re: [ccp4bb] to what extent bacterial expression reveals the native oligomerization state of mammalian proteins.

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

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Subject: Re: protein monitoring
From: Anastassis Perrakis a {- dot -} perrakis {- at -} NKI {- dot -} NL
Date: 2010-05-29

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Date: 2010-05-29

Subject: Re: to what extent bacterial expression reveals the native oligomerization state of mammalian proteins.
From: Anastassis Perrakis a {- dot -} perrakis {- at -} NKI {- dot -} NL
Date: 2010-05-29

Hi -

did you express your secreted mammalian protein in the E.coli
periplasm? If so, and if the oligomerization state is well defined, eg
its a 4mer, 5mer or whatever, and not a mixed population, I would be
quite confident that this is the 'true' native state. But, I would
only be sure in the presence of a functional assay: is your bacterial-
made protein functional? Thats the main question you need to ask. I do
not think that literature can help you establish anything here, its a
case dependent issue.

Having said all these, a well behaved protein produced in E.coli
(stable soluble, monodisperse),
is most likely in a native state, and I would go ahead with
experiments, e.g. crystallization while checking if its functional ...
But, before attempting to publish, you need to prove its functional,
and ideally prove the relevance of oligomerization by mutagenesis
experiments of the interface as soon as you get the structure.

Finally, mammalian expression systems for secreted mammalian proteins,
are really good, easy to use, and relatively cheap due to the
widespread use of growth media from biologists, and your institute is
likely to have most of the equipment you need, in the cell biology
department.

Check out these (open access) papers:

A time- and cost-efficient system for high-level protein production in
mammalian cells.Aricescu AR, Lu W, Jones EY. Acta Crystallogr D Biol
Crystallogr. 2006 Oct;62(Pt 10):1243-50.

Eukaryotic expression: developments for structural proteomics.
Aricescu AR, et al and , Jones EY. Acta Crystallogr D Biol
Crystallogr. 2006 Oct;62(Pt 10):1114-24.A.

On May 28, 2010, at 23:02, Jerry McCully wrote:

>
> A little update on my own project:
>
> It is a secreted protein without any modification reported. Size
> exclusion shows that it is an oligomer. CD spectrum shows well-
> defined secondary structure.
>
> It is stable and soluble.
>
> It may have different states in diseases' condition.
>
> Now I was trying to figure out the native state of this protein. As
> told by folks on CCP4bb, Most likely, the bacterial expression
> reveals its original state.
>
> Am I right?
>
> Thanks a lot,
>
> Jerry
> Date: Fri, 28 May 2010 11:40:31 -0700
> From: for-crystallizaiton@HOTMAIL.COM
> Subject: [ccp4bb] to what extent bacterial expression reveals the
> native oligomerization state of mammalian proteins.
> To: CCP4BB@JISCMAIL.AC.UK
>
> Dear ALL:
>
> I am sorry for this stupid question.
>
> I guess bacterial expression system is still most popular in
> structural biology.
>
> If you get a very good soluble E.coli expression of a human
> protein without disulfide bonds,
> to what extent do you believe that the oligomerization state of this
> bacterial expression will reflect the real physiological state of
> this protein in humans?
>
> Can someone give comments or refer some literature?
>
> Thanks a lot,
>
> Jerry McCully
>
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P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

CCP4bb navigation

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

Previous message:
Subject: Re: protein monitoring
From: Anastassis Perrakis a {- dot -} perrakis {- at -} NKI {- dot -} NL
Date: 2010-05-29

Next message:
Subject: Re: please recommend a crystallization incubator
From: Kris Tesh kris {- dot -} tesh {- at -} ATT {- dot -} NET
Date: 2010-05-29