I am working on thermus thermophilus protein which is not being expressed. I have tried that particular protein induction in a different time scale (12-37 degree), different IPTG concentration (o.1-1mM), and different strain (BL-21,Rosetta and c-43) but there is still no protein induction. What other methods could I try to induce my protein?
How do you know there is no expression? Did you make a western blot? Did you check the plasmid?
Check your plasmid sequence, has this protein been expressed using the same expression plasmid before as well?
How do you detect the protein expression? Do you use just a simple SDS gel? western blot?
@all....
I confirmed expression by western and SDS-PAGE both way but no induction.
Hi Ajeet,
You should then check your sequence in your plasmid. Be sure that your gene is within the coding frame. Also, what kind of expression plasmid are you using? You may want to employ some fusion tags to your protein (i.e.: MBP, SUMO; NUS A and etc) to aid in expression and folding/solubility of your protein.
More informations about your gene/plasmid would help to give the best advice.
Good Luck!!
First thing I would suggest is to retransform your plasmid into fresh BL21 competent cells and try again. Then if still negative reconfirm the sequence of the actual plasmid stock you used for the transformation.
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