Periplasmic production of pernisine in Escherichia coli and determinants for its high thermostability.

Periplasmic production of pernisine in Escherichia coli and determinants for its high thermostability. Appl Microbiol Biotechnol. 2020 Jul 30;: Authors: Bahun M, Hartman K, Poklar Ulrih N Abstract Pernisine is a subtilisin-like serine proteinase secreted by the hyperthermophilic archaeon Aeropyrum 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 of Escherichia 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 in E. coli to identify the minimally active domain. The catalytic domain of pernisine consists of the αẞα structurally similar core flanked by the N-terminal and C-terminal outer regions. The deletion of the C-terminal α helix did not affect the pernisine activity at 90 °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 °C. These results underline the importance of the Ca2+ binding sites predicted in these outer regions for stability and activity of pernisine. ...
Source: Applied Microbiology and Biotechnology - Category: Microbiology Authors: Tags: Appl Microbiol Biotechnol Source Type: research