Construction and expression of mutated variants of the Saccharomyces cerevisiae yeast SCW4 gene

Protein Scw4 veže se kovalentno i nekovalentno na staničnu stjenku kvasca Saccharomyces cerevisiae, no nije poznato na koji se način kovalentno veže. U potrazi za regijom potencijalno odgovornom za vezanje napravljena je delecijska analiza gena SCW4. Mutacije u konstruktima uvodene su PCR metodom megapočetnice koristeći plazmid pBG1805 SCW4 kao DNA kalup. U ovom radu napravljena su i ispitana dva delecijska konstrukta, Δ202SCW4 i Δ231SCW4, u kojima su deletirani dijelovi gena koji kodiraju za dio sekvence proteina od signalne sekvence do 202.-og, odnosno 231.-og aminokiselinskog ostatka. Konstrukti su ligirani u kvaščev plazmidni vektor YEp351 te su dobivenim plazmidima transformirane stanice kvasca. Kvasci su uzgajani u selektivnoj podlozi za uzgoj u uvjetima indukcije GAL1 promotora s obzirom da na to da GAL1 promotor na plazmidu YEp351 kontrolira ekspresiju mutiranih inačica SCW4 gena. Nakon uzgoja provedena je izolacija proteina stanične stijenke, proteini su razdvojeni u gelu SDS-elektroforezom po Laemmli-u te je pomoću Western blot metode potvrĎena ekspresija i nekovalentno vezanje mutiranog Scw4 proteina na staničnu stijenku kvasca.The Scw4 protein can be bound covalently or non-covalently to Saccharomyces cerevisiae yeast cell wall, but how the covalent linkage occurs is unknown. Searching for a region potentially responsible for the covalent binding, different constructs of the SCW4 gene were made. Mutations in gene were introduced by PCR megaprimers method using the pBG1805 SCW4 plasmid as template DNA. In this research two constructs were made and tested: Δ202SCW4 and Δ231SCW4. The constructs were made by the deletion of gene parts encoding 202 aminoacids (Δ202SCW4) or 231 aminoacids (Δ231SCW4), respectively, starting from the Scw4 signal sequence. Constructs were ligated into a yeast plasmid vector, YEp351 and introduced into the yeast cells. The transformed yeast cells were cultivated in a selective growth medium in conditions in which GAL1 promoter is induced, since the GAL1 promoter on YEp351 plasmid controls the expression of mutated variants of the SCW4 gene. After the cultivation, proteins were isolated from the yeast cell wall and then separated using the Laemmli gel SDS-electrophoresis. Expression and non-covalent binding of the mutated Scw4 proteins to yeast cell wall was confirmed by Western blot method