protein degradation in E coli .. help please

[gturcatel]

Hi all !!

I have big trouble expression my protein in E coli.
my protein is a DNA modifying enzyme, and I believe that is the problem : its toxicity.

The DNA seq is correct. I also add one extra stop codon (TAA) after the original one which is an TAG.

I used different e coli lines, also the rosetta2 .

I used induction at 37 C different times and RT up to over night.

I also tried to incubate the bacteria in protease inhibitors to avoid protein degradation.
any idea ?
thank you in advance for ur help
G.

[Kneeanderthal]

What kind of system are you using for induction? IPTG? If so then try lowering the IPTG to 0.2 mM. Also try doing the induction at a lower temperature (as low as 15 C) to get better expression. If the protein is toxic I would also recommend waiting until the culture has reached a high density (OD600 of 0.9 to 1.2) and then doing induction as it will increase the overall yield/per liter.

PS: If using IPTG add glucose to the media to further suppress the promoter before induction