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.
