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Applied and Environmental Microbiology, February 2004, p. 961-966, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.961-966.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Viral Preprotoxin Signal Sequence Allows Efficient Secretion of Green Fluorescent Protein by Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe

Antje Eiden-Plach, Tatjana Zagorc,{dagger} Tanja Heintel,{ddagger} Yvonne Carius, Frank Breinig, and Manfred J. Schmitt*

Angewandte Molekularbiologie, Universität des Saarlandes, D-66041 Saarbrücken, Germany

Received 9 September 2003/ Accepted 30 October 2003

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, and Schizosaccharomyces 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 (PGK1 and TPI1) or from inducible and/or repressible (GAL1, AOX1, and NMT1) 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.

* Corresponding author. Mailing address: Angewandte Molekularbiologie (FR 8.3), Universität des Saarlandes, Gebäude 2, Postfach 15 11 50, D-66041 Saarbrücken, Germany. Phone: (49) 681-3024730. Fax: (49) 681-3024710. E-mail: mjs{at}microbiol.uni-sb.de.

{dagger} Present address: Chamber of Commerce and Industry of Slovenia, SI-1504 Ljubljana, Slovenia.

{ddagger} Present address: Medizinische Mikrobiologie und Hygiene (Abteilung Virologie), Universität des Saarlandes, D-66421 Homburg/Saar, Germany.

Applied and Environmental Microbiology, February 2004, p. 961-966, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.961-966.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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