protein expression in E.coli - no expression after induction (Jul/28/2008 )

We use the pTrcHis TOPO TA cloning kit from Invitrogen to clone a mouse apolipoprotein. There was no expression after 1mM IPTG was added. Even with the expression control, we could only detect a very thin band on Commassie blue after the induction, which was confirmed by Western. We tried to slow down the growth rate of the bacteria by reducing the temperature and shaking speed. We have double checked almost everything we used such as IPTG, LB, ampicillin... and are pretty sure that they are OK. I have no idea if the protein is toxic, but apparantly its growth rate is the same as the negative control without insert and the expression control. We will really appreciate if anyone can help us.

Tianl

Dear Tainl,
1. I suggest checking your gene of interest for the presence of rare codons which can lead to poor expression.
2. I presume ur gene is in frame in the vector as u got faint signal by western. ( I presume u got the w blot signal at the expected size)

Regards
Avinash
http://aviprem.gq.nu