Pernisine is a subtilisin-like alkaline endopeptidase from the hyperthermophilic archaeon Aeropyrum pernix, known for its exceptional thermostability and ability to degrade prions. Like bacterial subtilisin, pernisine is synthesized in the archaeon as an inactive precursor and requires the assistance of an N-terminal pro-sequence (proregion) for folding into the mature enzyme. In subtilisin, the proregion functions not only as an inhibitor that blocks the active site prior to cleavage, but also as a molecular chaperone that enables correct folding of the catalytic domain. In contrast, the folding of pernisine is facilitated by Ca²⁺ ions, which bind to eight sites on the catalytic domain, whereas subtilisin folding is entirely dependent on the chaperone-like function of the proregion. Established methods for recombinant synthesis of pernisine in Escherichia coli and Streptomyces rimosus without the proregion have shown that the enzyme retains high thermostability and activity towards simple substrates. In this thesis, we investigated whether, in addition to Ca²⁺ ions, the proregion also influences the conformation, stability, and activity of the catalytic domain of pernisine. We successfully synthesized pernisine with the proregion in E. coli, whereas synthesis without the proregion was unsuccessful. Therefore, we focused on in vitro refolding of activated pernisine with and without the proregion after prior denaturation. Using various methods such as differential scanning fluorimetry, circular dichroism spectroscopy, fluorescence emission spectrometry, and degradation of a complex substrate, we analyzed the conformation, stability, and proteolytic activity of both in vitro refolded variants. The proregion did not show a significant influence in any of the analyses, indicating it does not affect any of the aforementioned properties. The results confirm previous assumptions that the proregion functions solely as an inhibitor and not as a chaperone for the catalytic domain.
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