Caspase-1 is a cysteine-aspartate protease that plays a key role in the inflammatory response by enabling the maturation of the pro-inflammatory cytokines interleukin-1β and interleukin-18 and participating in pyroptosis. Due to its important role in the development of numerous diseases, caspase-1 represents an important target for the development of new active pharmaceutical ingredients. The aim of this master’s thesis was to prepare recombinant human caspase-1 for evaluating potential selective inhibitors synthesized at the Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana. Two expression plasmids were prepared: the first contained the coding sequence for the p10 subunit of human caspase-1, while the second contained the coding sequence for the p20 subunit. Commercially available human caspase-1 complementary DNA was used as the starting material, from which the coding sequences for both subunits were amplified. Following successful ligation into the pET22b plasmid, the pET22b/p10 and pET22b/p20 vectors were used for transformation of the cloning strain E. coli TOP10. The constructs were confirmed by Sanger sequencing and subsequently used for transformation of the expression strain E. coli BL21-CodonPlus. Three experiments for the expression and isolation of recombinant caspase-1 were performed using two protocols. In the first experiment, following protein purification using the ÄKTA preparative chromatography system, the presence of the protein in the selected fractions was not confirmed by western blot analysis using specific antibodies. In the second and third experiments, despite various approaches to process optimization, soluble and enzymatically active caspase-1 could not be obtained due to difficulties with protein aggregation. Because the isolation was unsuccessful, a commercially available recombinant enzyme was used to evaluate the inhibitors. The reference inhibitor NSC697923 exhibited inhibitory activity in the nanomolar range, whereas the compound KEM-31A, synthesized at the Faculty of Pharmacy, inhibited caspase-1 in the low micromolar range. KEM-31A demonstrated promising inhibitory activity and represents a suitable lead compound for further optimization of potential caspase-1 inhibitors. In addition, the groundwork was established for further optimization of the recombinant human caspase-1 preparation process.
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