Bacterium Streptomyces rimosus is used in industry for production of antibiotic oxytetracycline (OTC) and therefore has GRAS (Generally Recognized as Safe) status. A number of proteins are secreted in medium. Therefore, the bacterium Streptomyces rimosus was tested for production of heterologous proteins, where gene encoding thermostable serine protease caminisine originally isolated from A. camini was integrated into S. rimosus genome. Amino acid composition of caminisine is very similar to pernisine from A. pernix, which was recently produced in S. rimosus. We designed several plasmid construced based on integrative plasmid pAB04, which included srt signal sequence, histidine tag, strong constitutive promoter ermE* and codon optimized DNA sequences for: mature caminisine (pAB04 ermE* srT Per AC CO HT), procaminisine (pAB04 ermE* srT ProPer AC CO HT) and preprocaminisine (pAB04 ermE* srT PreProPer AC CO HT). Based on azocasein assay and cPCR (colony PCR), we selected potentially interesting clones secreting recombinant caminisine for isolation with affinity chromatography. By applying SDS-PAGE, zymography and Western-blot assays, we confirmed the presence of different versions of recombinant pernisine. The amount of recombinant enzyme isolated with affinity chromatography was low and in addition few native were observed on the SDS-PAGE gels in the elution fractions. The highest specific proteolitic activity among different versions of the recombinant caminisine was observed for fully processed recombinant caminisine produced by S. rimosus ΔOTC transformed with pAB04 ermE* srT Per AC CO HT. Finally, it can be concluded that S. rimosus is potentially useful host for production of caminisine, however additional optimization of host strain and protein isolation methods would be needed.
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