Modifications of β-glucuronidase as a reporter gene for protein sorting in saccharomyces cerevisiae

Abstract

Several carboxypeptidase Y-/3-glucuronidase (CPY-GUS) plasmids were expressed in yeast. The CPY-GUS fusion proteins were inactivated during passage through the secretory pathway. This work was done in an attempt to recover GUS activity in yeast. CPY-GUS plasmids were transformed to protease-deficient yeast strains so as to test whether the low GUS activity was due to the degradation by intracellular proteases. The results failed to show this because of the problems associated with the western blots. Tunicamycin, a drug that inhibits glycosylation, was added to the yeast culture in order to recover GUS activity. The results show that a short term treatment could increase GUS activity by about 10%. In a long term treatment, tunicamycin was found to affect the normal growth of yeast adversely and reduce the GUS activity by 90%. Site-directed in vitro mutagenesis was employed to remove the two cryptic N-glycosylation sites within GUS. Two single mutants (160Mu and 16lMu) and one double mutant (l60+161)Mu were created. They were cloned in E.coli TG2 and their enzymatic activities were tested. The results show that all the mutated GUS (160Mu, 161Mu and (160+161Mu)) had higher enzymatic activities than the wild type GUS.