TANDEM OVERPRODUCTION AND CHARACTERIZATION OF THE NUCLEASE DOMAIN OF COLICIN E9 AND ITS COGNATE INHIBITOR PROTEIN IM9

We report the overproduction of the non-specific endonuclease domain o f the bacterial toxin colicin E9 and its preliminary characterisation in vitro. The enzymatic colicins (61 kDa) are normally released from p roducing cells in a complex with their cognate inhibitors, known as th e immunity proteins (9.5 kDa). Tryptic digestion of the purified ColE9 complex was found to generate two major components, a monomer derived from the N-terminal and central regions of the toxin and a heterodime r comprising the catalytically active C-terminal domain of the colicin bound to its intact immunity protein, Im9. N-terminal amino acid sequ encing, in conjunction with electrospray mass spectrometry, shows that preparations of the DNase domain isolated by this method are heteroge neous, thus making subsequent mechanistic and structural analysis diff icult. This problem was circumvented by selectively overexpressing the C terminal 15-kDa nuclease domain of colicin E9 in tandem with its co gnate inhibitor in Escherichia coli. This tandem overexpression strate gy allowed high-level production of a 25-kDa protein complex comprisin g the C-terminal DNase domain of colicin E9 tightly bound to its speci fic inhibitor Im9, thus masking the anticipated toxicity of the nuclea se. The DNase domain was then separated from Im9 under denaturing cond itions, refolded by removal of the denaturant and the renatured protei n shown to possess both endonuclease and Im9 binding activity. These r esults describe a novel method for the overproduction of a nuclease in bacteria by co-expressing its specific inhibitor and lay the foundati ons for a full mechanistic, biophysical and structural characterizatio n of the isolated DNase domain of the colicin E9 toxin.