Cathepsin S is a cysteine protease with a uniquely restricted expression in immune cells, where it participates in the endolysosomal protein degradation and MHC class II-mediated antigen presentation. Additionally, it plays an important role in cell death, inflammatory signaling in the cytoplasm, and various signaling and degradation pathways that take place in the extracellular milieu. Since cathepsin S is stable at a wide pH range, has wide substrate specificity and is subsequently highly proteolytically active in various cell compartments, its dysregulation can result in the development of various pathological conditions associated with chronic inflammation such as cancer, autoimmune and cardiovascular diseases and lung and bone diseases. As such it represents a promising therapeutic and diagnostic target, yet for its effective use, we need a thorough knowledge of its physiology first. Different natural and synthetic binding partners that can specifically bind cathepsin S and thereby enable its identification, visualization and quantification in in vitro and in vivo models, can be of great importance in cathepsin S research. Libraries containing small molecules or macromolecules can be screened using in vitro methods, whereby the tested molecules bind to an immobilized target protein - most commonly a biotinylated protein bound to a column with adhered (strept)avidin.
The aim of our work was to prepare a murine cathepsin S fused with the 15 amino acids long peptide sequence GLNDIFEAQKIEWHE, known as AviTag, since the latter enables site-specific biotinylation of the target protein using enzyme BirA. The sequence of the murine procathepsin S with AviTag was prepared using FX-cloning. The recombinant protein was expressed in Escherichia coli Rosetta-gamma B (DE3) pLysS and isolated by Ni2+-chelate affinity chromatography. After activation, the concentration of the active enzyme was assessed by titration of the active site of the enzyme with the irreversible inhibitor E-64. We showed that lowering the pH of the activation solution to 4,5, addition of DTT and as little as a 10-15 minute incubation of the enzyme at 37 ° C is sufficient for its complete activation. With prolonged incubation the enzyme self-degradation occurs, resulting in protein loss. Despite considerable losses of the active murine cathepsin S with AviTag we were able to prepare sufficient quantity of it, which allows us to continue with site-specific biotinylation of the protein and subsequent selection of specific binding partners in vitro.
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