expression problem! - (May/17/2007 )

Hi all,

in the last weeks I've been trying (obviously unsuccessfully ) to express cholera toxin subunit B in e.coli. The gene is inserted in a plasmid for expression in vibrio sp. 60. But i do not have the "bugs". The cloning worked well in e. coli and then i said let's give it a try. I tried 3 different temperatures 37; 30 and 20 in combination with 2 different induction times 3 and 18 hours. For induction i used IPTG 1mM. And started the induction once at OD₆₀₀=1.2 once at OD=0.6 and once OD=0.2.
What else can i do?
Any suggestion is more then welcome!!

Thanks

Chemx

PS: i had to work with what i found in the lab, and there are not many things there ....

what are the results of your experiments? Do you notice any drop in OD after induction? What stain of e coli are you using?

strain B834 (DE3), and no drop in OD after induction. Simply i see no protein production when i compare to the control (empty vector)...

No drop in OD. Meaning it is not toxic towards E.coli. How about the sequence? Is it in frame?

What method did you use for cell lysing to get the protein?

Cholera toxin B subunit (CTB) is not toxic to E. coli. Our lab has already expressed CTB it in E. coli with no problems, except that the pentamer doesn't always form (i.e. there are some monomers present that havent pentamerized or the pentamers degraded during electrophoresis).

I had problems with IPTG induced expression of my protein in E. coli. After changing every conceivable condition, I figured out that IPTG stock solutions break down quickly, especially after freeze-thaw cycles. I used fresh IPTG powder and added it directly to growing cultures, then my expression went up dramatically.

Hope this helps!

1st: i used chemolysis of my cells, i think it worked looking at the gel...(at least i hope so!)

2nd: well, my IPTG stock was quite "fresh" one or 2 weeks old. but adding powder IPTG , this sounds nice, I'll try it!

Thanks!

1st: i used chemolysis of my cells, i think it worked looking at the gel...(at least i hope so!)

2nd: well, my IPTG stock was quite "fresh" one or 2 weeks old. but adding powder IPTG , this sounds nice, I'll try it!

Thanks!