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Expression, processing and high level secretion of a virus toxin in fission yeast
T Heintel1, T Zagorc, M J Schmitt
1Angewandte Molekularbiologie, Universität des Saarlandes, Saarbrücken, Germany.
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.
Insights
Schizosaccharomyces pombe can express the K28 killer toxin, demonstrating functional processing and secretion. This fission yeast shows potential for enhanced heterologous protein expression and secretion.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Protein Secretion
Background:
- Saccharomyces cerevisiae K28 toxin kills sensitive cells via cell cycle arrest.
- The toxin is processed from preprotoxin (pptox) to an active alpha/beta heterodimer.
- Schizosaccharomyces pombe lacks natural toxin-secreting killer strains.
Purpose of the Study:
- To investigate if Schizosaccharomyces pombe can express the K28 killer toxin.
- To assess the processing and secretion efficiency of K28 toxin in Schizosaccharomyces pombe.
- To evaluate Schizosaccharomyces pombe as a host for heterologous protein expression.
Main Methods:
- Constructed K28 pptox gene cassettes for Schizosaccharomyces pombe.
- Utilized thiamine-regulated expression under the Sch. pombe nmt1 promoter.
- Performed Northern and Western blot analyses to detect transcript and protein.
Main Results:
- Schizosaccharomyces pombe successfully expressed a functional K28 killer phenotype.
- The toxin was correctly processed and secreted by fission yeast.
- Toxin secretion levels were at least 10-fold higher than in Saccharomyces cerevisiae.
Conclusions:
- Schizosaccharomyces pombe is capable of expressing and secreting functional K28 toxin.
- Fission yeast offers a highly efficient system for heterologous protein secretion.
- Pptox-derived vectors show promise for biotechnological applications.