Protein with 13 Prolines

Frank Küster frank at kuesterei.ch
Thu Dec 4 04:56:00 EST 2003


D.K. <dk at no.email.thankstospam.net> schrieb:

> On Tue, 02 Dec 2003 11:04:44 +0100, frank at kuesterei.ch (Frank Küster) wrote:
>>senmehmetsen at yahoo.com (Mehmet Sen) schrieb:
>>
>>> There are also 4
>>> cysteine, making s-s bonds. 
>>
>>Hm, and possibly PDI with the right GSH-GSSG cocktail.
>
> Just couple questions here, Frank: 
>
> I am about to embark on my first "serious" refolding effort
> for crystallization purposes. I found literature both 
> disparate and overwhelming. 

I'm probably not the best person to ask, because I'm concentrating on
proteins that fold easily and fast, to investigate the
mechanism. However, especially in my Ph.D. thesis I was unlucky enough
to make some experiences with misfolding proteins...

> Any hints on finding out
> what "right" GSH-SSSG cocktail is? Also, what is the usual 
> source for PDI? Do people buy it or express themselves? 

I would first look for papers of Rainer Rudolph from Halle,
Germany. (Rudolph-R[au] & halle[ad] in medline). Reviews of him in
Medline are rather old, unfortunately. But he has published a couple of
papers on refolding of disulfide bonded human proteins. There must also
be some papers of Rudi Glockshuber (ETH Zürich), but he concentrates
less on the biotechnological aspects, rather on elucidating the detailed
mechanism. 

As for the source, I don't know (E.coli DsbA?) - but I know people who
know and can ask them if you like.

> Just how serious is the improvement upon PDI addition 
> (success rate or yield)? 

As far as I remember, the question is whether you have multiple SS-bonds
or only one. With multiple SS-bonds, but without PDI you get all SS
scrambled under oxidizing conditions, and the yield of native protein is
really low. If you refold at nearly reducing conditions, they can
rearrange, but folding (or rather oxidation) is very slow then. E.coli
DsbA and oxidizing conditions should give better yields, because
oxidation is fast then, but the PDI allows for fast rearrangement of the
wrong disulfides.

Bye, Frank
-- 
Bitte daran denken: Auch die Kaffeekasse hat manchmal Hunger.



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