Madsen Claus Krogh, Vismans Gilles, Brinch-Pedersen Henrik
Department of Molecular Biology and Genetics, Section for Crop Genetics and Biotechnology, Aarhus University, Forsogsvej 1, 4200 Slagelse, Denmark.
Wageningen University, 6708PB Wageningen, The Netherlands.
J Microbiol Methods. 2016 Oct;129:1-7. doi: 10.1016/j.mimet.2016.07.015. Epub 2016 Jul 18.
The methylotrophic yeast Pichia pastoris is a popular host for recombinant expression of proteins. Plasmids containing the Pichia autonomously replicating sequence (PARS) transform P. pastoris with higher efficiency than linear DNA equipped with termini designed for homologous recombination. Moreover, PARS containing constructs provide higher protein yields. Unfortunately, these autonomous plasmids are inherently unstable and the preferred method of P. pastoris transformation is therefore stable integration in the genome by homologous recombination. In the present study we report that a novel combination of PARS and linearization of plasmids for P. pastoris transformation serves to significantly increase the transformation efficiency. Moreover, it is demonstrated that the constructs do not re-circularize but integrate stably into the P. pastoris genome.
甲基营养型酵母巴斯德毕赤酵母是用于蛋白质重组表达的常用宿主。含有毕赤酵母自主复制序列（PARS）的质粒转化毕赤酵母的效率高于配备用于同源重组的末端的线性DNA。此外，含有PARS的构建体可提供更高的蛋白质产量。不幸的是，这些自主质粒本质上是不稳定的，因此毕赤酵母转化的首选方法是通过同源重组稳定整合到基因组中。在本研究中，我们报告了一种用于毕赤酵母转化的PARS与质粒线性化的新组合，可显著提高转化效率。此外，还证明了构建体不会重新环化，而是稳定整合到毕赤酵母基因组中。
