Scorer C A, Clare J J, McCombie W R, Romanos M A, Sreekrishna K
Department of Cell Biology, Wellcome Research Laboratories, Beckenham, Kent, UK.
Biotechnology (N Y). 1994 Feb;12(2):181-4. doi: 10.1038/nbt0294-181.
Pichia pastoris is a methylotrophic yeast increasingly important in the production of therapeutic proteins. Expression vectors are based on the methanol-inducible AOX1 promoter and are integrated into the host chromosome. In most cases high copy number integration has been shown to be important for high-level expression. Since this occurs at low frequency during transformation, we previously used DNA dot blot screens to identify suitable clones. In this paper we report the use of vectors containing the Tn903 kanr gene conferring G418-resistance. Initial experiments demonstrated that copy number showed a tight correlation with drug-resistance. Using a G418 growth inhibition screen, we readily isolated a series of transformants, containing progressively increasing numbers (1 to 12) of a vector expressing HIV-1 ENV, which we used to examine the relationship between copy number and foreign mRNA levels. Northern blot analysis indicated that ENV mRNA levels from a single-copy clone were nearly as high as AOX1 mRNA, and increased progressively with increasing copy number so as to greatly exceed AOX1 mRNA. We have also developed protocols for the selection, using G418, of high copy number transformants following spheroplast transformation or electroporation. We anticipate that these protocols will simplify the use of Pichia as a biotechnological tool.
巴斯德毕赤酵母是一种在治疗性蛋白质生产中日益重要的甲基营养型酵母。表达载体基于甲醇诱导型AOX1启动子，并整合到宿主染色体中。在大多数情况下，高拷贝数整合已被证明对高水平表达很重要。由于这种情况在转化过程中发生频率较低，我们之前使用DNA斑点杂交筛选来鉴定合适的克隆。在本文中，我们报告了使用含有赋予G418抗性的Tn903 kanr基因的载体。初步实验表明，拷贝数与耐药性密切相关。通过G418生长抑制筛选，我们很容易分离出一系列转化体，其中含有表达HIV-1 ENV的载体，其数量逐渐增加（1至12个），我们用这些转化体来研究拷贝数与外源mRNA水平之间的关系。Northern印迹分析表明，单拷贝克隆的ENV mRNA水平几乎与AOX1 mRNA一样高，并随着拷贝数的增加而逐渐增加，从而大大超过AOX1 mRNA。我们还开发了在原生质体转化或电穿孔后使用G418选择高拷贝数转化体的方案。我们预计这些方案将简化毕赤酵母作为生物技术工具的使用。
