Viral preprotoxin signal sequence allows efficient secretion of green fluorescent protein by Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe
| Autor: | Antje Eiden-Plach, Tanja Heintel, Manfred J. Schmitt, Frank Breinig, Yvonne Carius, Tatjana Zagorc |
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| Rok vydání: | 2004 |
| Předmět: |
Signal peptide
Saccharomyces cerevisiae Green Fluorescent Proteins Candida glabrata Protein Sorting Signals Applied Microbiology and Biotechnology Pichia Pichia pastoris Green fluorescent protein Fungal Proteins Gene Expression Regulation Fungal Yeasts Schizosaccharomyces Protein Precursors Ecology biology Mycotoxins biology.organism_classification Physiology and Biotechnology Yeast Killer Factors Yeast Recombinant Proteins Culture Media Luminescent Proteins Biochemistry Schizosaccharomyces pombe Food Science Biotechnology |
| Zdroj: | Applied and environmental microbiology. 70(2) |
| ISSN: | 0099-2240 |
| Popis: | Besides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata,Pichia pastoris,Saccharomyces cerevisiae, andSchizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1andTPI1) or from inducible and/or repressible (GAL1,AOX1, andNMT1) yeast promoters. In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast. |
| Databáze: | OpenAIRE |
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