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 <[EMAIL PROTECTED]>: > 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 >> > > > **NOTA DE CONFIDENCIALIDAD** Este correo electrónico, y en su caso los > ficheros adjuntos, pueden contener información protegida para el uso > exclusivo de su destinatario. Se prohíbe la distribución, reproducción o > cualquier otro tipo de transmisión por parte de otra persona que no sea el > destinatario. Si usted recibe por error este correo, se ruega comunicarlo al > remitente y borrar el mensaje recibido. > **CONFIDENTIALITY NOTICE** This email communication and any attachments may > contain confidential and privileged information for the sole use of the > designated recipient named above. Distribution, reproduction or any other use > of this transmission by any party other than the intended recipient is > prohibited. If you are not the intended recipient please contact the sender > and delete all copies. > -- ============================ David C. Briggs PhD Father & Crystallographer http://drdavidcbriggs.googlepages.com/home AIM ID: dbassophile ============================
