Pernisine is a subtilisin-like serine proteinase secreted by the hyperthermophilic archaeonAeropyrum pernix. The significant properties of this proteinase are remarkable stability and ability to degrade the infectious prion proteins. Here we show the production of pernisine in the periplasm ofEscherichia coli. This strategy prevented the aggregation of pernisine in the cytoplasm and increased the purity of the isolated pernisine. The thermostability of this recombinant pernisine was significantly increased compared with previous studies. In addition, several truncated pernisine variants were constructed and expressed inE. colito identify the minimally active domain. The catalytic domain of pernisine consists of the alpha & x1e9e;alpha structurally similar core flanked by the N-terminal and C-terminal outer regions. The deletion of the C-terminal alpha helix did not affect the pernisine activity at 90 degrees C. However, the complete deletion of the C-terminal outer region resulted in loss of proteolytic activity. The pernisine variant, in which the N-terminal outer region was deleted, had a reduced activity at 90 degrees C. These results underline the importance of the Ca(2+)binding sites predicted in these outer regions for stability and activity of pernisine.