Cathepsin B is a cysteine peptidase belonging to the papain-like peptidase group. It is a monomeric and globular enzyme present in most living organisms, participating in various fundamental physiological and pathological processes. In this study, we attempted to prepare a dimeric version of cathepsin B. Oligomerization of enzymes can, in certain cases, lead to changes in their properties, such as increased stability or greater catalytic efficiency. Therefore, we attempted to mimic this process in laboratory experiments. Initially, we selected 19 positions in the amino acid chain on the lower surface of the protein, according to the standard orientation, to create mutant libraries using the site-specific saturation mutagenesis method. We screened the prepared libraries for dimerization using bacterial adenylate cyclase based two-hybrid system, identifying 15 potential dimers with mutations at 6 different positions in the amino acid chain. We attempted to express and isolate 4 potential dimers, succeeding in only one case. We analyzed a dimer composed of variants catB_SNS154,156WNH and catB_SNS154,156GNP using size-exclusion chromatography. We found that under the conditions in which the experiment was conducted, no dimerization occurred. We activated the variants autocatalytically. The enzyme activity of the isolated variants was confirmed using kinetic methods and we determined their Km values which were and 34±6 µM in 142±54 µM respectively.
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