Viral Preprotoxin Signal Sequence Allows Efficient Secretion of Green Fluorescent Protein byCandida glabrata,Pichia pastoris,Saccharomyces cerevisiae, andSchizosaccharomyces pombe

Abstract

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.Over the years, heterologous protein expression in yeast species such as Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe has become an important tool in the production of therapeutic and commercially relevant proteins. The advantage of a yeast-based expression system is due to the fact that as a eukaryotic microorganism, yeast combines ease of genetic manipulation and cell growth with a well-known capacity to perform complex posttranslational protein modifications (4,22,29). Because yeast per se secretes only low levels of endogenous proteins, a secreted recombinant protein usually contributes significantly to the total amount of proteins in the culture medium. Thus, directing a foreign protein for secretion is an important initial step in its subsequent purification.While the vast majority of heterologous proteins has been expressed within the cytosol of the corresponding host, only a few proteins have been successfully secreted into the extracellular medium. In most eukaryotic proteins, the critical initial step in protein secretion is their co-and/or posttranslational translocation into the lumen of the endoplasmic reticulum (ER) followed by subsequent sorting into the Golgi network. Foreign protein import into the ER is usually achieved by fusing the protein of interest in frame to a homologous secretion signal sequence derived from a naturally secreted protein of the corresponding host, thus conferring secretion competence to the desired protein fusion (13,23).In yeast, the most widely used secretion sign...

Expression of GFP using Pichia pastoris vectors with zeocin or G‐418 sulphate as the primary selectable marker

“…Used in combination, therefore, these two vectors would permit simultaneous transformation and expression of two heterologous genes; which would provide a powerful tool for the expression and purification of heterodimeric proteins, such as members of the integrin superfamily (Berman et al, 2003). Analogous constructs with the pPICZα and pPICKanMX6α vectors could express a secretable form of the GFP reporter protein, in accordance with previous observations (Campbell and Choy, 2002;Eiden-Plach et al, 2004). The ability to express recombinant proteins in P. pastoris using our G-418 sulphate resistance vectors under conditions of high resistance stringency, and the subsequent purification of the product by relatively simple chromatographic procedures, thus extends the practical utility of this host cell system for the generation of recombinant proteins.…”

Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm

“…The work was carried out with E. coli ATCC 33456 pEGFP, a strain that forms a robust biofilm and harbored a high copy number modified pUC vector encoding green fluorescent protein (GFP). GFP has been used in diverse systems to model HPP, including yeast (Cha et al, 2004;Eiden-Plach et al, 2004), bacteria (Belanger et al, 2004;Reischer et al, 2004), mammalian cells (Condon et al, 2003;Hu et al, 2003), insects (Kramer et al, 2003), and plants (Borisjuk et al, 1999;Komarnytsky et al, 2000). By comparing plasmid maintenance and GFP expression by E. coli ATCC 33456 pEGFP grown in both the chemostat and as a biofilm, it was possible to determine whether the biofilm environment positively influenced HPP.…”

Novel Yeast Bioassay for High-Throughput Screening of Matrix Metalloproteinase Inhibitors

“…In an initial attempt to validate eukaryotic yeasts as hosts for the expression of human MMPs, we screened various biotechnologically relevant species, such as Kluyveromyces lactis, P. pastoris, S. cerevisiae, Schizosaccharomyces pombe, and Zygosaccharomyces bailii, for efficient processing and secretion of human proMMP-2 and proMMP-9. In each case, a species-specific Nterminal signal peptide was used to ensure proMMP import into the yeast secretory pathway (2,4). Successful secretion of human MMPs into the culture supernatant was verified by Western analysis and zymography, indicating that P. pastoris was the only yeast capable of producing and secreting human MMPs in a biologically active form and at a constant and sufficiently high quality (unpublished results; see Fig.…”