subcloning - expression problem (Mar/01/2005 )

I tried to clone a fragment into an expression vector, but everytime it fails , i tried the mock cloning, it works but the one with the insert does not grow, or i just get a grow without the fragment. i have been told that the insert might be a toxin one and killing the host , any comments and how to rectify this.

thank you.

if your assumtion is, that the peptide derived from your DNA-Insert in your vector is toxic, you might want to consider using another expression system, which uses are tighter regulation of expression. If toxicity is a problem, it should manifest itself only after induction of expression, not in clonig itself, and when it does, it's a sign on a leaky promotor.

Another possible way is to alter your peptide by fusing it to GST, for example, or expression of only parts, preferably known domains of your protein, seperately of each other to avoid toxic products.

mike

Thank you mike , il try the modification and see, thankyou again.