The protease caminisine, isolated from the archaea Aeropyrum camini, belongs to the group of subtilisin-like proteases. Its amino acid sequence is similar to the previously characterised protease pernisine from the archaea Aeropyrum pernix, which can degrade prion proteins. Due to the low yields and extreme growth conditions of A. camini, large-scale production of the protease caminisine is not feasible. In the framework of the MSc thesis, the bacterium Streptomyces rimosus was used as a heterologous host for the production of an engineered caminisine complex with the fluorescent protein mCherry. Constructed plasmid vectors for the production of engineered proteins caminisine, pro- caminisine, mCherry-caminisine and mCherry-pro-caminisine in S. rimosus (pAB04_ermE*_srT_kaminizin / pro-kaminizin / mCherry-kaminizin / mCherry-pro-kaminizin_AC CO HT) were transformed by electroporation into S. rimosus ΔOTC, which has the gene cluster encoding oxytetracyline biosynthesis deleted. The transformants were then grown in different media and the extracellular activities of the constructed fusion proteins were compared. The presence of mCherry was determined by fluorescence measurements and the activity of caminisine variants was determined by an azo-casein assay. We demonstrated a correlation between fluorescence intensity and caminisine activity. After isolation of the enzyme from the supernatant by TCA precipitation or nickel-affinity chromatography, the expression efficiency of the recombinant caminisine was assessed by NaDS-PAGE and the proteolytic activity was confirmed by cimography and the azo-casein assay. Affinity chromatography failed to significantly concentrate the caminisine from the supernatant, which was reflected by the low intensity of the protein bands on the NaDS-PAGE gel. Total extracellular proteins isolated by TCA were visualised on NaDS-PAGE gels and on a cimogram to verify the effect of prior temperature activation on protein content. We demonstrated that the mCherry-caminisine fusion protein has the same proteinase activity as caminisine alone. The effect of different temperatures, pH values and NaDS concentrations on the activity of caminisine was also tested. We found that the highest activity of caminisine as a fusion protein is highest at 80 °C and pH 8,0.
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