I've had a band in the region you describe when expressing GST tagged
proteins, and ID'd it with mass spec.

I seem to recall it was a "FKB-type peptidyl-prolyl-cis-trans-isomerase."
http://www.uniprot.org/uniprot/P45523




2008/10/17 Mazzorana.Marco <[log in to unmask]>:
> Dear all,
> may I point out at this common belief that the 'GST' Kumar wrote about is probably not GST?
>
> When trying to over-express my protein in fusion with a GST tag I have noticed that many strains did not express it, but still a large band corresponding 25 kDa protein  was visible from the gel. The amount of this protein is IPTG concentration dependent and also time dependent, but it doesn't stick to Gluthatione-sepharose beads as GST should. I guess that the protein is not GST at all. Does anybody have any clue of what's exactly going on (i.e. does anyone know which protein we are talking about)?
>
> Concerning the original question, I would suggest to try other strains. My recent experience with a GST-protein let me obtain a huge amount of the fusion product from the old JM109, rather than from the more common BL21 and derivatives (such as BL21 star and B834).
>
> Ciao,
>
>
> Marco
>
>
>
>
>
>
>
> -----------------------------------------------------------
> Marco Mazzorana, PhD
>
> Grupo de Cristalografía de Macromoléculas
>
> CNIO - Centro Nacional de Investigaciones Oncológicas
> C/ Melchor Fernández Almagro, 3, E-28029 Madrid (España)
>
> Phone: +34 91 2246985
> Fax: +34 91 2246976
> -----------------------------------------------------------
>
>
>
> -----Original Message-----
> From: CCP4 bulletin board on behalf of Mark J. van Raaij
> Sent: Fri 17/10/2008 14.59
> To: [log in to unmask]
> Subject: Re: [ccp4bb] GST fusion-Expression problem
>
> Depending on your exact question, I can see the following possible
> problems:
> - a stop codon has been introduced after the GST, either by a cloning
> error or spontaneous mutation (if the fusion protein is toxic, these
> sponateous mutations may be relatively frequent). Are you using
> glycerol stocks for your starter culture? Try using a fresh
> transformation from the day before to minimise introduction of
> spontaneous mutations. In fact, I never use glycerol stocks, and
> always use fresh transformations, after finding out this problem the
> hard way and losing valuable time.
> - the fusion protein is unfolded/toxic/otherwise problematic, and
> proteases chew it away during expression. Try growing the culture, and
> also the starter culture, also at 20 ºC also before induction. In
> combination, also try induction with low concentrations of IPTG (0.01
> mM or less) to ensure plasmid-bearing cells stay viable and are not
> outgrown by others.
> - or, if expression of the fusion protein is ok, but you get only GST
> and little of the eukaryotic fusion partner upon protease cleavage, it
> may be that the partner is poorly folded and only kept in solution by
> the GST. Here, a possible solution would be an eukaryotic expression
> system.
>
> Mark J. van Raaij
> Dpto de Bioquímica, Facultad de Farmacia
> Universidad de Santiago
> 15782 Santiago de Compostela
> Spain
> http://web.usc.es/~vanraaij/
>
>
>
>
>
>
>
> On 17 Oct 2008, at 14:39, Putcha, Balananda Dhurjati Kumar wrote:
>
>>
>> Dear CCP4ers,
>>
>> I have a GST fusion protein (eukaryotic).  When I express it in
>> E.coli BL21 (DE3) or RIPL cells, however, I mostly get GST and very
>> little fusion protein.  I get very little "leaky expression".  I
>> normally grow my cells at 37C in LB or 2X YT and after induction at
>> 20C for 16-18hrs.  I also have tried growing at 37C for 2hrs (after
>> induction) with little change.  Is there a trick to solving this
>> problem?   Thank you for your thoughts
>> Kumar
>>
>
>
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-- 
============================
David C. Briggs PhD
Father & Crystallographer
http://drdavidcbriggs.googlepages.com/home
AIM ID: dbassophile
============================