Kumar,
Sometimes a pGEX vector with thrombin site causes such a problem. Using TEV or HRV3C sites may help. Did you see any band corresponding to the protein without GST? A simple way to check expression is to induce at 37C for 0.5-2 hours and run a gel of whole cell lysate. If there is expression, you then figure out how to make the protein soluble. We had one case that lowering temperature diminished protein expression. Good luck! Chun _____ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Putcha, Balananda Dhurjati Kumar Sent: Friday, October 17, 2008 5:39 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] GST fusion-Expression problem 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
