Listeria monocytogenes is an intracellular pathogen, which can cause severe infections in humans and other vertebrates. It is found in soil, water, vegetation and food; most of infections in humans are caused by ingestion of contaminated food. One of the main virulence factors of the bacteria is phospholipase C (PC-PLC), which together with listeriolysin O enables the escape of bacteria from acidic phagosome into host cell cytosol, where it replicates and spreads into neighbouring cells. PC-PLC activity depends on three zinc atoms in the active site. The activity optimum is in acidic pH (5-6) and the highest affinity is towards phosphatidylcholine phospholipids. The aim of this master thesis was to study, how mutations in PC-PLC gene sequence effect PC-PLC enzymatic activity and binding on model lipid membranes in presence of zinc ions. In bacterial expression system seven mutated phospholipases PC-PLC genes were successfully expressed and mutant proteins synthesized. Mutant phospholipases had substitutions or deletions in tryptophan residue at the active site of protein (W1A, W1E, W1F, W1K, ?WS) and two cysteine mutant proteins (C143S, C168K and C143S, C168S). Proteins were purified by chitin affinity and gel chromatography. Isolated proteins were characterized using biochemical (SDS-PAGE, enzymatic activity assays, binding assay) and biophysical (circular dichroism) methods. Our results showed that mutations had in general no effect on the stability of PC-PLC, but they effected enzymatic activity and binding of protein on lipid membranes. It was shown that addition of zinc ions increased enzymatic activity and binding of PC-PLC on lipid membranes. With optimization of PC-PLC crystallography we established the basis for future determination of phospholipase's three-dimensional structure.
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