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Revista de la Sociedad Química del Perú

Print version ISSN 1810-634X

Abstract

MANSILLA GARCIA, Sandy Nelly  and  KITAZONO SUGAHARA, Ana Akemi. Determination of the secretion efficiency of a recombinant protein in the yeast S. cerevisiae by electrophoretic analysis and Western- Blot detection. Rev. Soc. Quím. Perú [online]. 2023, vol.89, n.3, pp.240-248.  Epub Mar 30, 2023. ISSN 1810-634X.  http://dx.doi.org/10.37761/rsqp.v89i3.434.

The main goal of this work was to investigate the secretion levels that can be achieved when using two different mechanisms in the yeast Saccharomyces cerevisiae and the enzyme glutathione S-transferase (GST), and via electrophoresis and Western-blotassays. The respective plasmids were previously constructed, which allowed the production of the GST enzyme fused to two different secretion signals (signal peptides, SP): the alpha SP (derived from the alpha mating factor) and a synthetic SP (designed from consensus sequences derived from proteins that are known to be efficiently secreted in yeast). After the intracellular and extracellular protein fractions were separated by electrophoresis, a Western blot assay was performed using a monoclonal anti-GST antibody for the specific detection of the protein. These assays allowed the identification of several intracellular forms of GST with diverse sizes depending on the SP, but only the mature 26 kDa form was found in the extracellular fraction. However, no difference was observed on the extracellular levels of GST, which indicated that, under the applied conditions, both SPs are similarly efficient promoting protein secretion.

Keywords : immunoblotting; glutathione S- transferase (GST); yeast Saccharomyces cerevisiae; secretion levels; enzyme secretion; alpha signal peptide; synthetic signal peptide.

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