One of the most common hospital-acquired infections is Clostridioides difficile infection, which causes severe and recurrent diarrhoea. Recently, infection with this bacterium has also been associated with cases of colitis. The pathogenicity of the bacterium is derived from toxins A and B, but colonisation of the gut is also a key step. The S-layer proteins are key to this process. The most important S-layer protein is surface layer protein A (SlpA), which homogeneously builds the entire S-layer. Even more important is the self-assembly process of this layer, which involves the cysteine protease Cwp84. Cwp84, an 84 kDa membrane enzyme, cleaves SlpA into two subunits, one with a low molecular weight (LMW) and the other with a high molecular weight (HMW), before starting to assemble the S-layer. Spore-forming bacteria are almost inaccessible to the immune system in the human body, so it is important to investigate ways that could inhibit the proper maturation of the S-layer and its self-assembly. We assumed that inhibition of the cysteine protease Cwp84 could be used for the development of antimicrobial agents in combination with antibiotics. Therefore, as part of my master thesis, we wanted to express a fragment of the Cwp84 enzyme containing a catalytic and a lectin-like domain. To this end, we included the notation of Cwp84 enzyme fragment in two vectors, one of which had a GST fusion tag that would help us to properly fold the enzyme and isolate it. Despite several attempts, we have not been able to express and quantify the kinetic properties of the Cwp84 enzyme.
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