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Saccharomyces cerevisiae is able to sense and respond to environmental changes such as the availability of carbon sources. In a previous work, we showed that the expression of the Pre S2- S gene of HBV in yeast was negatively regulated at the translational level dependent of glucose. In this study, we show that the S m RNA is detected in the polysomes indicating its active translation, while the Pre S2- S m RNA was mainly found in monosomes. Moreover, we used the gene reporter assay based on Zeocin resistance, to better characterize the Pre S2 region responsible for this control. Two chimeric genes composed of the N- and C-terminal part of the Pre S2 fused to the Sh-bleomycin gene conferring the resistance to Zeocin were expressed in yeast. We found that the strain expressing the N-terminal part of the Pre S2 was sensitive to Zeocin on rich medium with 2% glucose. In contrast, the strain harbouring the C-terminal part of the Pre S2 fused to the Sh-bleomycin grew on Zeocin, indicating that the Sh-bleomycin m RNA is efficiently translated, subsequently conferring resistance to Zeocin. Our data suggest the establishment of a translational control via the N-terminal part of the Pre S2 mediated by the presence of 2% glucose in the media. [ABSTRACT FROM AUTHOR]

The yeasts Saccharomyces cerevisiae and Pichia pastoris are attractive hosts for production of human proteins. The main advantages offered by these systems are the well-developed and easily accessible genetic tools, rapid growth, the simple and inexpensive culture media, and many of the cellular and metabolic processes found in higher eukaryotes are conserved in both yeast species. In this chapter, we describe the production of two proteins of therapeutic interest: the human P53 tumor suppressor and the viral HBsAg in P. pastoris and S. cerevisiae using the strong and inducible promoters AOX1 and Gal10/Cyc1, respectively. Besides the production as a goal of both expressions, we also report on an unexpected result that has occurred in S. cerevisiae: The overexpression of human p53 induces yeast cell death with characteristic markers of apoptosis, such as the externalization of phosphatidylserines and DNA strand cleavage.

In the present study, we have compared the expression level of the genes encoding the viral Hepatitis B S and preS2 using two different yeast vectors, a constitutive one (YepIPT) and a galactose-inducible one (YepDP1-8). We showed that the S and preS2 mRNAs were present in equivalent amounts in both systems, while the corresponding proteins showed a different pattern. The S and preS2 proteins were efficiently produced after galactose induction. However, under the constitutive promoter, the S protein was synthesized at the same level in presence of 2% glucose or galactose whereas the preS2 protein was efficiently synthesized on galactose but absent on 2% glucose. The substitution of glucose by non-repressive carbon sources such as 2% galactose, ethanol or glycerol led to a significant expression of preS2. A high level of preS2 expression was also achieved by lowering the glucose concentration. Our data suggest that glucose exerts a concentration dependent negative translational control on the preS2 mRNA.