Real-time PCR-based determination of gene copy numbers in Pichia pastoris
Real-time PCR-based determination of gene copy numbers in Pichia pastoris
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DOI：
10.1002/biot.200900233
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发表时间：
2010-04-01
影响因子：
4.7
通讯作者：
Glieder, Anton
中科院分区：
文献类型：
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作者：
Abad, Sandra;Kitz, Kerstin;Glieder, Anton
巴斯德毕赤酵母是用于异源蛋白生产的优选宿主。表达盒通常整合到该甲基营养型酵母的基因组中。本文描述了一种快速、可靠地测定巴斯德毕赤酵母表达菌株基因拷贝数的方法。我们认为，基因拷贝数的测定对于所有研究巴斯德毕赤酵母的研究人员以及许多其他使用类似基因整合技术转化其他酵母的研究小组都很重要。所述方法使用实时PCR来定量整合的表达盒。类似的方法以前用于其他宿主系统，如动物和植物细胞，但没有这样的方法比较不同的检测方法和描述细节的酵母分析，通过定量PCR是我们已知的，特别是甲基营养型酵母，如巴斯德毕赤酵母。忽略基因拷贝数可以容易地导致对来自密码子优化或启动子研究的实验结果的错误解释以及辅助蛋白的共表达，如在本文中也描述的应用实例中所证明的。
Pichia pastoris is a preferred host for heterologous protein production. Expression cassettes are usually integrated into the genome of this methylotrophic yeast. This manuscript describes a method for fast and reliable gene copy number determinations for P. pastoris expression strains. We believe that gene copy number determinations are important for all researchers working with P. pastoris and also many other research groups using similar gene integration techniques for the transformation of other yeasts. The described method uses real-time PCR to quantify the integrated expression cassettes. Similar methods were employed previously for other host systems such as animal and plant cells but no such method comparing different detection methods and describing details for yeast analysis by quantitative PCR is known to us, especially for methylotrophic yeasts such as P. pastoris. Neglecting gene copy numbers can easily lead to false interpretations of experimental results from codon optimization or promoter studies and co-expression of helper proteins as demonstrated in an application example, which is also described here.