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Ptn expressed once... and never again!!! - (Mar/03/2009 )

I don't know how to explain this but I'm trying to express a 14KDa bovine recombinant protein clonned into a pET23a.

Using BL21 PLysS, I got an expression band (no western confirmed) using 0.5 mM IPTG. The picture of the gel is attached. (control was non-induced culture).

The problem is that I never got this expression band again! I re-utilized the same clone, I've transformed more bacteria.. but nothing works!!!

I wonder if it a gel artifact ... but the band is so clear.

Could you help me?!
Attached Image


Then, did you transform again with the expression construction on BL21 cells?. You know that you should┤t store the plasmid inside the expression strain, you should always store it in another non-expression strain such us Xl1-blue, isolate from here and transform the expression strain, then perform a screening of expression clones. I have noticed that when you store your expression construction in a expression strain you can loose much of your expression, i guess due to appearance or selection of mutants which were able to get rid of the expression of the heterologous protein, mainly if it is toxic for bacteria.
Maybe you can reduce the temperature at which you perform the expression experiments, for example, you can change from 37║C to RT or reduce even more the IPTG to 0.1 mM.