Modulation of the catalytic activity of a metallonuclease by tagging with oligohistidine.

Peptide tags are extensively used for affinity purification of proteins. In an optimal case, these tags can be completely removed from the purified protein by a specific protease mediated hydrolysis. However, the interactions of these tags with the target protein may also be utilized for the modulation of the protein function. Here we show that the C-terminal hexahistidine (6 × His) tag can influence the catalytic activity of the nuclease domain of the Colicin E7 metallonuclease (NColE7) used by E. coli to kill competing bacteria under stress conditions. This enzyme non-specifically cleaves the DNA that results in cytotoxicity. We have successfully cloned the genes of NColE7 protein and its R447G mutant into a modified pET-21a DNA vector fusing the affinity tag to the protein upon expression, which would be otherwise not possible in the absence of the gene of the Im7 inhibitory protein. This reflects the inhibitory effect of the 6 × His fusion tag on the nuclease activity, which proved to be a complex process via both coordinative and non-specific steric interactions. The modulatory effect of Zn2+ ion was observed in the catalytic activity experiments. The DNA cleavage ability of the 6 × His tagged enzyme was first enhanced by an increase of metal ion concentration, while high excess of Zn2+ ions caused a lower rate of the DNA cleavage. Modelling of the coordinative effect of the fusion tag by external chelators suggested ternary complex formation instead of removal of the metal ion from the active center.