Treatment of the infection with Clostridioides difficile bacterium represents a major problem in the modern world due to its resistance to antibiotics. Infections occur especially in hospitalized patients treated with antibiotics which cause dysbiosis of the intestinal microbiota. C. difficile causes inflammation and bleeding from the large intestine (colitis), which leads to severe diarrhea and may even result in death. The major virulence factors of C. difficile are the TcdA and TcdB exotoxins, glucosyltransferases that act on GTPases from the Rho family, thereby weakening tight and adherense junctions in the mucosal epithelium. Recombinant probiotics represent a bioalternative antimicrobial approach for the treatment of C. difficile infection and are the subject of many (non)clinical studies. C. difficile infection could be reduced or prevented by a combination of bacteriophage endolysins, selective for C. difficile, and specific protein binders of TcdA and TcdB toxins, expressed on the surface of the recombinant probiotic bacterium Lactococcus lactis. In the diploma thesis, we focused on obtaining toxin binders (from the group of affimers and DARPins; alone or in fusion with the C-terminal domain of the anchor protein AcmA, which enables binding to the cell wall of L. lactis) and endolysins using the Escherichia coli expression system. In the first part of the thesis, we prepared expression vectors: we inserted synthetic genes for selected proteins into the modified plasmid pET28c (pET28c-HTF). We transformed the expression strain E. coli NiCo21(DE3) with the plasmids and performed test expressions of recombinant proteins after induction with IPTG, and qualitatively assessed the amount of recombinant products by sodium dodecyl sulfate polyacrylamide gel electrophoresis. We found that exotoxin binders in fusion with AcmA are not soluble. Based on the results we selected only the soluble proteins (i.e. exotoxin binders without AcmA and endolysins) for scale-up expression. In the second part of the thesis we isolated recombinant proteins using metal-chelate affinity chromatography and concentrated them by ultrafiltration. We found that fusion proteins of TcdA/TcdB exotoxin binders with the AcmA anchor domain are present in the insoluble fraction of the lysate and are deposited in inclusion bodies under the expression conditions used.
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