3 replies to this topic

[ouhscr2]

Dear All,

My protein of interest is 296 a.a (MW 34.2kD). If I over express the full length of this protein it causes E. coli cell to die and little protein induction. However, if I over express either the C and N Terminus of this protein, we get nice induction. We tried with couple of E. coli strains to over express it and got the same results.
Thus, I would like to know what it is the reason behind this phenomenon.
Experimental conditions:
E. coli: C41(DE3) also BL21(DE3)
Plasmid: pET-15b
IPTG: 1mM
Temp: 37C
Growth medium: 2xYT
OD: before induction 0.6

Any clue, please advice.

Thank you.

[Carmela]

Hi!
I have similar problems with my proteins...
You can try different temperatures. Are you doing the expression at 37ºC? You could try growing and expression at 28, or growing at 37 and expressing at 25 or even 16. I had success with the last combination...

[mdfenko]

if the full length protein is killing the bacteria then it is toxic. you may want to try expressing in a different organism (yeast?).

[Carmela]

Another idea: try a vector with periplasmic signal... pET...¿16?¿40? I'm not sure right now... but for toxic proteins could be an alternative (I avoid yeast when I can)