Lysins are hydrolytic enzymes encoded by bacteriophages that degrade bacterial cell wall during the lytic cycle of bacteriophage reproduction. The goal of this master thesis was to heterologously express lysins, that would display lytic activity against Propionibacterium acnes and Staphylococcus epidermidis. These bacteria are amongst the causative agents of nocosomial infections and different non-lethal medical conditions in humans. We used polymerase chain reaction (PCR) to isolate genes encoding for lysins from bacteriophages infecting these 2 bacteria. In the case of P. acnes, we could not obtain a full-length lysin gene from a P. acnes bacteriophage E1 so we did not express any P. acnes lysins in our work. On the other hand, we successfully isolated 3 lysins from a staphylococcal bacteriophage (StphK), namely 114, 154 and 156. We cloned the lysin genes into 2 plasmid vectors using recombinant DNA methods: pQE-80L in pMAL-C5X. Next, we transformed the vectors containing inserts into E. coli strains Top10 and Er2523. In order to obtain mutation-free lysin genes, we sequenced the inserts we isolated from transformant colonies (clones). In total, we sequenced 13 clones of lysins in 2 vectors. Clones with the minimal number of mutations were used for expression. Phusion polymerase did not provide a lower mutation rate than Taq polymerase, despite its proofreading ability. Expression of lysins was successful in pMAL-C5X but not in pQE-80L. We found that pMAL-154 exhibited autoproteolytic activity and that pMAL-114 was proteolytically degraded. Lysins were partially purified to crude form (cell lysates). Lysins were found to be present in both the soluble and insoluble fraction with the solubility test. To determine lytic activity of crude lysins, we used the plate count assay. None of the lysins exhibited any lytic activity against the staphylococcal strain SeC2.
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