I have a clone of my protein coding ORF in pGEX-4T-1 vector which should produce a fusion protein of ~115kDa. Snapgene Analysis of the clone (sequence) shows that the clone is in proper orientation. However, even after repeated induction trials, I don't see any fusion protein expression although, GST is expressed in the control (Vector pGEX-4T-1 only). I have tried out with 0.1, 1, 3, 5 mM IPTG, at 37 deg C for 1, 2, 4, 6, 8, 12, 24 hrs. Can anyone help me troubleshoot this?
Am badly in need for this !!