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 !!
Hi there,
Did you check the sequence is in the same frame for both fusion partners and that there is no unwanted stop codon?
Do you mean there is no expression at all or that the protein is expressed as an insoluble matter?
The sequence is in frame. I don't see any expression in the PAGE in the probable zone of MW.
Hi again, did you have a look at the insoluble? Did you probe with anti-GST? Expression might be low and undetectable by gel staining. If it's about to improve expression then different paraeters might be optimized: temperature, OD for induction, length of induction, autoinduction media...
But if there is no expression at all I would suggest to switch for an other system.
Hi Rohit,
May I ask what bacterial host you are using? Also, have you tried codon optimization to increase the expression efficiency?
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