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.
Key points
• Aggregation of produced pernisine was prevented by translocation into periplasm.
• Thermostability of mature pernisine was increased.
• The outer regions of the catalytic core are required for pernisine thermostability.
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Acknowledgments
We are thankful to Marko Dolinar for providing the pMD204 plasmid.
Funding
This work was supported by the Slovenian Research Agency through grant L7-8277 (to NPU) and postgraduate research funding (to MB). We are also thankful to Borer Chemie AG for financial support through the L7-8722 grant.
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MB and NPU designed research. MB and KH conducted experiments. MB analyzed data and wrote the manuscript. NPU contributed materials/ analytical tools and coordinated the project. All authors read and approved the manuscript.
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Bahun, M., Hartman, K. & Poklar Ulrih, N. Periplasmic production of pernisine in Escherichia coli and determinants for its high thermostability. Appl Microbiol Biotechnol 104, 7867–7878 (2020). https://doi.org/10.1007/s00253-020-10791-w
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DOI: https://doi.org/10.1007/s00253-020-10791-w