3 replies to this topic

[chn09]

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

[swanny]

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.

[chn09]

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

[sitting_at _the _AKTA]

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