Department of Biotechnology, BOKU, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
FEMS Yeast Res. 2009 Dec;9(8):1260-70. doi: 10.1111/j.1567-1364.2009.00561.x. Epub 2009 Aug 6.
The yeast Pichia pastoris is a widely used host organism for heterologous protein production. One of the basic steps for strain improvement is to ensure a sufficient level of transcription of the heterologous gene, based on promoter strength and gene copy number. To date, high-copy-number integrants of P. pastoris are achievable only by screening of random events or by cloning of gene concatemers. Methods for rapid and reliable multicopy integration of the expression cassette are therefore desirable. Here we present such a method based on vector integration into the rDNA locus and post-transformational vector amplification by repeated selection on increased antibiotic concentrations. Data are presented for two exemplary products: human serum albumin, which is secreted into the supernatant, and human superoxide dismutase, which is accumulated in the cytoplasm of the cells. The striking picture evolving is that intracellular protein production is tightly correlated with gene copy number, while use of the secretory pathway introduces a high clonal variability and the correlation with gene copy number is valid only for low gene copy numbers.
酵母毕赤酵母是一种广泛用于异源蛋白生产的宿主生物。菌株改良的基本步骤之一是确保异源基因的转录水平足够，这取决于启动子强度和基因拷贝数。迄今为止，只有通过随机事件筛选或基因串联克隆才能获得毕赤酵母的高拷贝数整合子。因此，需要快速可靠的多拷贝整合表达盒的方法。本文提出了一种基于载体整合到 rDNA 位点和转化后通过在增加的抗生素浓度下反复选择进行载体扩增的方法。本文提供了两个示例产物的数据：人血清白蛋白，它分泌到上清液中，和人超氧化物歧化酶，它积累在细胞的细胞质中。逐渐呈现出的惊人结果是，细胞内蛋白质的产生与基因拷贝数密切相关，而使用分泌途径会引入很高的克隆变异性，并且与基因拷贝数的相关性仅适用于低拷贝数。
