Expression, processing and high level secretion of a virus toxin in fission yeast.
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.
Heintel
T
T
Angewandte Molekularbiologie, Universität des Saarlandes, Saarbrücken, Germany.
Zagorc
T
T
Schmitt
M J
MJ
D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D005822:Genetic Vectors; D055646:Killer Factors, Yeast; D009183:Mycotoxins / Q000096:biosynthesis* / Q000235:genetics; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000235:genetics*; D013831:Thiamine / Q000494:pharmacology
C527157:KHR1 protein, S cerevisiae; D055646:Killer Factors, Yeast; D009183:Mycotoxins; D011498:Protein Precursors; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D013831:Thiamine