Viral preprotoxin signal sequence allows efficient secretion of green fluorescent protein by candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe
Viral preprotoxin signal sequence allows efficient secretion of green fluorescent protein by candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe
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DOI：
10.1128/aem.70.2.961-966.2004
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发表时间：
2004-02-01
影响因子：
4.4
通讯作者：
Schmitt, MJ
中科院分区：
文献类型：
--
作者：
Eiden-Plach, A;Zagorc, T;Schmitt, MJ
除了作为真核细胞生物学中的模式生物的重要性之外，酵母物种还已经发展成为用于表达、加工和分泌重组蛋白的有吸引力的宿主。在这里，我们研究了外源蛋白分泌在四个远亲酵母（光滑念珠菌，巴斯德毕赤酵母，酿酒酵母和裂殖酵母粟酒裂殖酵母）通过使用绿色荧光蛋白（GFP）作为报告和病毒分泌信号序列来自K28 preprotoxin（pptox），酵母K28病毒毒素的前体。与N-末端pptox前导序列融合的GFP的体内表达和/或整个pptox基因的表达由组成型（PGK 1和TP 11）或诱导型和/或阻遏型（GAL 1、AOX 1和NMT 1）酵母启动子驱动。在每种情况下，GFP进入相应宿主细胞的分泌途径;共聚焦荧光显微镜以及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和无细胞培养上清液的Western分析证实GFP被有效地分泌到培养基中。除了用GFP观察到的结果之外，全长病毒pptox在所有四种酵母属中被正确加工，导致生物活性病毒毒素的分泌。综上所述，我们的数据表明，病毒K28 pptox信号序列有可能被用作重组蛋白生产中的独特工具，以确保在酵母中有效的蛋白分泌。
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 TP11) 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.