High level expression of His-tagged colicin pore-forming domains and reflections on the sites for pore formation in the inner membrane.

There exists ample evidence for the assumption that pore-forming colicins cannot exert their toxicity within the producing cell and that they must gain access to the outer face of the cytoplasmic membrane to achieve this. We wished to construct pET-vectors to produce pore-forming domains of colicin A and N with N-terminal hexa-histidine tags under the control of a T7 promoter. This was only possible when the correct immunity protein was also present. Hence it appears that this system exhibits the peculiarity that there is a toxicity associated with the over produced pore-forming domain. However, when the ratio of colicin to immunity protein is compared it is still clear that direct insertion into the cytoplasmic membrane does not occur and that membrane translocation of the colicin at limited sites may be occurring. This article reviews previous literature on the subject in terms of a model for limited sites of colicin action.