Overexpression of binding protein and disruption of the PMR1 gene synergistically stimulate secretion of bovine prochymosin but not plant Thaumatin in yeast
Overexpression of binding protein and disruption of the PMR1 gene synergistically stimulate secretion of bovine prochymosin but not plant Thaumatin in yeast
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DOI：
10.1007/s002530050831
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发表时间：
1996-11-01
影响因子：
5
通讯作者：
Maat, J
中科院分区：
文献类型：
--
作者：
Harmsen, MM;Bruyne, MI;Maat, J
当异源蛋白索马甜和牛凝乳酶原在酵母细胞中与酵母转化酶分泌信号肽融合产生时，不到2%的产物以生物活性形式分泌到培养基中。其余部分以错误折叠构象积聚在细胞内。我们研究了是否可以通过结合蛋白（BiP）的过度表达来改善这种不良的分泌，BiP是真核细胞中的主要伴侣之一。事实上，由于将额外的 kar2 基因拷贝引入含有单个整合拷贝的转化酶/凝乳酶原融合基因的酵母细胞中，结合蛋白的水平增加了 10 倍，导致胞外凝乳酶原的量增加了 20 倍以上。通过额外破坏这些细胞的 PMR1 基因，我们能够分泌几乎所有产生的凝乳酶原。另一方面，结合蛋白过度表达并没有显着刺激索马甜的输出。
When the heterologous proteins thaumatin and bovine prochymosin are produced in yeast cells as a fusion with the yeast invertase secretory signal peptide, less than 2% of the product is secreted in a biologically active form into the medium. The remainder accumulates intracellularly in a misfolded conformation. We investigated whether this poor secretion can be improved by overexpression of binding protein (BiP) one of the major chaperones in eukaryotic cells. Indeed, a tenfold increase in the level of binding protein, as a result of the introduction of extra copies of the kar2 gene into yeast cells containing a single, integrated copy of the invertase/prochymosin fusion gene, caused more than a 20-fold increase in the amount of extracellular prochymosin. By additional disruption of the PMR1 gene of these cells we were able to obtain secretion of virtually all of the prochymosin produced. Export of thaumatin, on the other hand, was not significantly stimulated by binding protein overexpression.