Re: [ccp4bb] GST fusion-Expression problem

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

Previous message:
Subject: Re: Twinning for P31
From: Eleanor Dodson ccp4 {- at -} YSBL {- dot -} YORK {- dot -} AC {- dot -} UK
Date: 2008-10-17

Next message:
Subject: Re: GST fusion-Expression problem - NOT GST
From: David Briggs drdavidcbriggs {- at -} GMAIL {- dot -} COM
Date: 2008-10-17

Subject: Re: GST fusion-Expression problem
From: "Mazzorana {- dot -} Marco" mmazzorana {- at -} CNIO {- dot -} ES
Date: 2008-10-17

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: CCP4BB@JISCMAIL.AC.UK
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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CCP4bb navigation

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

Previous message:
Subject: Re: Twinning for P31
From: Eleanor Dodson ccp4 {- at -} YSBL {- dot -} YORK {- dot -} AC {- dot -} UK
Date: 2008-10-17

Next message:
Subject: Re: GST fusion-Expression problem - NOT GST
From: David Briggs drdavidcbriggs {- at -} GMAIL {- dot -} COM
Date: 2008-10-17