Harmsen M M, Bruyne M I, Raué H A, Maat J
Department of Biochemistry and Molecular Biology, IMBW, BioCentrum Amsterdam, Vrije Universiteit, The Netherlands.
Appl Microbiol Biotechnol. 1996 Nov;46(4):365-70. doi: 10.1007/BF00166231.
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.
当在酵母细胞中与酵母转化酶分泌信号肽融合产生奇异果甜蛋白和牛凝乳酶原这两种异源蛋白时，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的构象在细胞内积累。我们研究了真核细胞中主要伴侣蛋白之一结合蛋白（BiP）的过表达是否能改善这种分泌不佳的情况。实际上，通过将kar2基因的额外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，使结合蛋白水平提高了十倍，导致细胞外凝乳酶原的量增加了20倍以上。通过对这些细胞的PMR1基因进行额外破坏，我们能够使几乎所有产生的凝乳酶原都得以分泌。另一方面，奇异果甜蛋白的输出并未因结合蛋白的过表达而受到显著刺激。
