Plasmid instability in E.coli BL21? - (Sep/04/2009 )
Im trying to express a bacterial protein in E. coli. Initaially the protein was soluble following overexpression in E. coli BL21. Now i find that the protein is going into the insoluble fraction. Could this be due to some mutation in the plasmid or is it due to plasmid instability .pls help
-chn09-
chn09 on Sep 4 2009, 10:00 PM said:
Im trying to express a bacterial protein in E. coli. Initaially the protein was soluble following overexpression in E. coli BL21. Now i find that the protein is going into the insoluble fraction. Could this be due to some mutation in the plasmid or is it due to plasmid instability .pls help
What conditions are you running with? Media / temperatures / IPTG / etc.
I doubt the plasmid would be the cause, but you can check that with a re-sequence of the insert.
-swanny-
swanny on Sep 7 2009, 08:03 AM said:
chn09 on Sep 4 2009, 10:00 PM said:
Im trying to express a bacterial protein in E. coli. Initaially the protein was soluble following overexpression in E. coli BL21. Now i find that the protein is going into the insoluble fraction. Could this be due to some mutation in the plasmid or is it due to plasmid instability .pls help
What conditions are you running with? Media / temperatures / IPTG / etc.
I doubt the plasmid would be the cause, but you can check that with a re-sequence of the insert.
Im growing the E. coli BL21 clone at 37 0C, and inducing it with 0.4 mM IPTG for a duration of 4 hrs
-chn09-
It's most likely the growing conditions of the bugs that have lead to the protein going into inclusion bodies. I express my proteins at 20 deg C to help keep them soluble. Making a fusion protein of your protein-of-interest to Maltose Binding protein can also help but this is a bit of a pain cause you'd have to make a new DNA construct
-sitting_at _the _AKTA-