bacterial growth arrest after IPTG induction - (Oct/06/2008 )

hi,

i am trying to express and purify His-tagged plant antifungal proteins in ecoli. I am using pET32 vector. The gene has been cloned inframe and confirmed with sequencing.


The probelm i am facing is after induction with IPTG(0.3to 0.6mM) the bacterial growth is getting completely arrested. there was no expression of my protein in cells collected at various intervals after induction.

Even after 24 hrs the culture exibit the same turbidity as it was before induction. where as uninduced culture is growing normally.

In literature similar proteins from various plants were previously expressed and purified from Ecoli.

Can any one suggest me what can be done to overcome this. Is there any possibility that this protein might having antibacterial activity leading to growth arrest??? or any optimisation of experiment which will help.

thanks a lot in advance...

Gene Tag

hi

As u notice it is possible that ur protein causes growth arrest or even decrease of the OD measurement.

This phenomenon is typical for toxic protein (usually membrane proteins), or proteins with high expression.

So what can u do, 1) be sure that u use suitable medium maybe rich medium would be preferable for ur culture growing.

2) did u try induction of the cells at lower temp. 17C ? its helps preventing fast growing of the cells and death.

thanks for your reply amtash.

I haven't tried yet the two options u suggested ..

i tried only temparatures ranging 24C to 30 C.

So i will give a trial.

thanks once again for ur suggestions...

cheers