Bibliographic citations
Mansilla, S., (2021). Clonación en plásmidos para la producción y secreción de péptidos o proteínas en Saccharomyces cerevisiae” [Universidad Nacional Agraria La Molina]. https://hdl.handle.net/20.500.12996/4690
Mansilla, S., Clonación en plásmidos para la producción y secreción de péptidos o proteínas en Saccharomyces cerevisiae” []. PE: Universidad Nacional Agraria La Molina; 2021. https://hdl.handle.net/20.500.12996/4690
@misc{renati/1113864,
title = "Clonación en plásmidos para la producción y secreción de péptidos o proteínas en Saccharomyces cerevisiae”",
author = "Mansilla García, Sandy Nelly",
publisher = "Universidad Nacional Agraria La Molina",
year = "2021"
}
The main goal of this thesis was to investigate the secretion of glutathione S-transferase (GST) and a few oligopeptides in Saccharomyces cerevisiae by cloning the respective genes in plasmids. The constructed plasmids included also the sequences coding for two types of signal peptides (PS): “PS-Alpha”, derived from the alpha mating factor and “PS-Synthetic”, designed from the consensus sequences for the PSs of the Yps1/Yap3/TA57 proteins. All plasmids were assembled in vivo, and the inserts were verified by analytical PCR and then by nucleotide sequencing. GST production and secretion were verified by Western blot assays, resulting in the detection of bands of the expected size (26 kDa) and others of larger size, depending on the signal peptide (PS) used. Interestingly, while the secretion efficiencies observed were similar for both PSs, in the case of PS-Synthetic the GST found intracellularly showed larger sizes, suggesting a greater amount of glycosylations. Likewise, a simple assay was designed to compare the photoprotective effects of peptides previously identified in our laboratory. The survivability of the cells transformed with the secretion plasmids was evaluated by irradiating them with UV. The results suggest a moderate photoprotective capacity but it is necessary to expand these studies. In conclusion, the S. cerevisiae homologous recombination machinery allows optimal cloning of inserts for plasmid construction. The GST protein can be secreted by using the signal peptides PS-Alpha and PS-Synthetic, but the last causes a greater number of modifications.
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