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Protein erilizin B je 59 kD velik protein iz užitne gobe kraljevi ostrigar (Pleurotus eryngii). Če ga rekombinantno izražamo v bakteriji Escherichia coli, se ne zvija pravilno in tvori inkluzijska telesca, posledično pa je doprinos topnega proteina zelo nizek. Erilizin B smo zato želeli izraziti v kvasovki Saccharomyces cerevisiae, saj smo pričakovali, da se bo protein izražal v topni obliki in da ga bomo izolirali v višjih koncentracijah. Izražanje rekombinantnega proteina erilizina B smo preverjali na več načinov. Spremljali smo hitrost rasti kvasovke S. cerevisiae in protein skušali zaznati s poliakrilamidno elektroforezo v prisotnosti natrijevega dodecil sulfata in s prenosom western. Ker je erilizin B v kombinaciji s proteinom ostreolizinom A6 hemolitičen, smo njegovo prisotnost preverili s hemolitičnim testom na govejih eritrocitih. V primeru, ko smo izražali erilizin B, označen z zelenim fluorescenčnim proteinom (EryB-eGFP), smo rast rekombinantnih kvasovk spremljali z merjenjem fluorescence in jih vzporedno opazovali s fluorescenčnim mikroskopom. Izražanja rekombinantnega proteina erilizina B nismo zaznali, zato sklepamo, da kvasovka S. cerevisiae v kombinaciji z ekspresijskim vektorjem pYES2 ni primeren ekspresijski sistem za pridobivanje rekombinantnega erilizina B. Erylysin B is a 59 kD protein from the king oyster mushroom (Pleurotus eryngii). When erylysin B is recombinantly expressed in Escherichia coli, it does not fold properly and forms inclusion bodies. The amount of soluble protein is therefore very low. The main goal of the MSc thesis was therefore to express erylysin B in the yeast Saccharomyces cerevisiae. We have expected the expression of recombinant protein in soluble form and in higher concentrations in contrast to the E. coli expression system. Therefore, the expression of the recombinant erylysin B was tested in several ways. We followed the growth curves of the transformed yeast cells and the protein expression was monitored using sodium dodecyl sulphate–polyacrylamide gel electrophoresis and Western blot analysis. Since erylysin B in the combination with ostreolysin A6 is haemolytic, its presence was also monitored by haemolytic assay on bovine erythrocytes. In the case when eGFP-labelled erylysin B was expressed, the fluorescence measurements were performed, as well as fluorescent microscopy in order to follow the expression of the protein in living cells. The expression of the recombinant erylysin B was not detected, so we concluded that the yeast S. cerevisiae in combination with the expression vector pYES2 is not a suitable expression system for obtaining recombinant erylysin B protein. |
