Expression, processing and high level secretion of a virus toxin in fission yeast

Abstract

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.

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

“…Heintel et al recently demonstrated that fission yeast is capable of secreting a correctly processed and biologically active K28 virus toxin after in vivo expression of the K28 pptox gene under transcriptional control of the thiamine-repressible nmt1 ϩ promoter (12). To investigate whether such plasmiddriven killer phenotype expression is also possible in P. pastoris and C. glabrata, episomal vectors were constructed in which the entire K28 pptox gene was expressed either from the constitutive PGK1 promoter (C. glabrata) or from the methanol-inducible AOX1 promoter (P. pastoris).…”

“…Escherichia coli DH5␣ cells [F Ϫ recA1 endA1 gyrA96 thi hsdR17 supE44 relA ⌬(argF-lacZYA) U169 80(dlacZ⌬M15) Ϫ ] grown in LuriaBertani medium were used as a host for the amplification and propagation of all constructed plasmids. Yeast cultures were grown at 30°C either on complex yeast extract-peptone-dextrose medium or on synthetic complete minimal medium containing 0.67% yeast nitrogen base (YNB) without amino acids supplemented with ammonium sulfate and the appropriate amino acid-base requirements of each strain (12,17). As indicated and previously described (9), the culture medium for methanol-induced pptox and/or GFP expression in P. (12,21).…”

“…In addition to the results seen with leader sequences derived from naturally secreted host cell proteins, Heintel et al recently showed that a viral secretion signal from the K28 killer virus is equally functional in baker's yeast and fission yeast, indicating that a preprotoxin (pptox)-based signal sequence might be a novel and unique tool for manipulating heterologous protein secretion in biotechnologically relevant yeast species (12). We now demonstrate-to our knowledge for the first time-that a viral pptox leader sequence is indeed capable of ensuring efficient green fluorescent protein (GFP) secretion in four distantly related yeast species (C. glabrata, P. pastoris, S. cerevisiae, and S. pombe), indicating that this signal sequence might be of general importance for foreign protein secretion in yeast.…”

“…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 signals are those derived from yeast invertase (Suc2p), acid phosphatase (Pho5p), inulinase (Inu1p), ␣-galactosidase (Mel1p), or pheromone ␣-factor or from the plasmid-driven killer toxin of Kluyveromyces lactis (6,12,16,23). In addition to the results seen with leader sequences derived from naturally secreted host cell proteins, Heintel et al recently showed that a viral secretion signal from the K28 killer virus is equally functional in baker's yeast and fission yeast, indicating that a preprotoxin (pptox)-based signal sequence might be a novel and unique tool for manipulating heterologous protein secretion in biotechnologically relevant yeast species (12).…”

“…In yeast, the most widely used secretion signals are those derived from yeast invertase (Suc2p), acid phosphatase (Pho5p), inulinase (Inu1p), ␣-galactosidase (Mel1p), or pheromone ␣-factor or from the plasmid-driven killer toxin of Kluyveromyces lactis (6,12,16,23). In addition to the results seen with leader sequences derived from naturally secreted host cell proteins, Heintel et al recently showed that a viral secretion signal from the K28 killer virus is equally functional in baker's yeast and fission yeast, indicating that a preprotoxin (pptox)-based signal sequence might be a novel and unique tool for manipulating heterologous protein secretion in biotechnologically relevant yeast species (12).…”