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...

The differential fractions of perforin-expressing virus-specific CD8 T cells in HIV and CMV double infection might be caused by differences in priming and trafficking to or from replication sites. However, without knowing the underlying mechanism, the fraction of perforin-expressing HIV-specific CD8 T cells provides another surrogate marker for disease progression.

The human cellular immune response against 14 distantly related yeast species was analyzed by intracellular cytokine staining of lymphocytes after ex vivo stimulation of whole blood. While the CD4 T cell response was marginal, extensive MHC class I-restricted CD8 T cell responses were detected against a number of species including spoiling, environmental and human pathogenic yeasts. The yeast-specific CD8 T cells expressed interferon-gamma but lacked expression of CD27 and CCR7, indicating that they were end-differentiated effector memory cells. Mainly intact yeast cells rather than spheroplasts were able to induce cytokine expression in T cells demonstrating that the dominant immunogens were located in the yeast cell wall. Together these data underline the importance of the cellular immune response in protecting humans against yeast and fungal infections. And, from another perspective, recombinant yeast suggests itself as a potential vaccine candidate to efficiently induce antigen-specific CD8 T cell responses.

The virally encoded K28 toxin of Saccharomyces cerevisiae kills sensitive yeast cells in a multi-step receptor-mediated fashion by cell cycle arrest and inhibition of DNA synthesis. In vivo, the toxin is translated as a 38 kDa preprotoxin (pptox) which is enzymatically processed to the biologically active alpha/beta heterodimer during passage through the yeast secretory pathway. Here, we demonstrate that Schizosaccharomyces pombe, a yeast from which no natural toxin-secreting killer strains are known, is perfectly capable of expressing a killer phenotype. Episomal as well as integrating K28 pptox gene cassettes were constructed that allowed a tightly thiamine-regulated killer phenotype expression under transcriptional control of the Sch. pombe nmt1 promotor. Northern analysis of the toxin-coding transcript as well as Western analysis of the secreted toxin indicated that fission yeast is capable of expressing a correctly processed and fully functional virus toxin. Moreover, toxin secretion in recombinant Sch. pombe was at least ten-fold higher than in any natural and/or recombinant Sac. cerevisiae killer strain, indicating that pptox-derived vectors might be attractive in the fast growing field of heterologous protein expression and secretion in yeast.

Threatening virus infections constantly illustrate the growing need for novel vaccines that specifically induce efficient T cell-mediated immune responses. In this study, we used a human whole blood assay to determine the activation of antigen-specific human T lymphocytes by a viral antigen of human cytomegalovirus (HCMV). The major HCMV tegument protein pp65, recombinantly expressed in fission yeast (Schizosaccharomyces pombe), specifically activated antigen-specific CD4- and CD8-positive memory T cells in blood of HCMV seropositive donors. Moreover, the immune response against recombinant pp65, in particular that of CD8 class I major histocompatibility complex-restricted cytotoxic T cells, was similar to the response against the intact HCMV. Since fission yeast cells per se did not activate a significant number of human T lymphocytes ex vivo, the system described here might represent a novel approach in vaccine development as well as in the identification of vaccine candidates directly from human whole blood.