Department of Life Sciences, Imperial College London, London, UK.
Centre for Synthetic Biology and Innovation, Imperial College London, London, UK.
Sci Rep. 2017 Nov 17;7(1):15817. doi: 10.1038/s41598-017-16172-0.
Although recent advances in E. coli self-assembly have greatly simplified cloning, these have not yet been harnessed for the high-throughput generation of expression strains in the early research and discovery phases of biopharmaceutical production. Here, we have refined the technique and incorporated it into a streamlined workflow for the generation of Pichia pastoris expression strains, reducing the timeline by a third and removing the reliance on DNA editing enzymes, which often require troubleshooting and increase costs. We have validated the workflow by cloning 24 human proteins of biopharmaceutical value, either as direct therapeutics or as research targets, which span a continuous range in size and GC content. This includes demonstrating the applicability of the workflow to three-part assemblies for a monoclonal antibody and its single-chain antibody fragments derivatives. This workflow should enable future research into recombinant protein production by P. pastoris and a synthetic biology approach to this industrial host.
尽管大肠杆菌自组装的最新进展极大地简化了克隆，但这些进展尚未在生物制药生产的早期研究和发现阶段被用于高通量表达菌株的生成。在这里，我们改进了该技术，并将其纳入毕赤酵母表达菌株生成的简化工作流程中，将时间缩短了三分之一，并消除了对 DNA 编辑酶的依赖，这些酶通常需要故障排除并增加成本。我们通过克隆 24 种具有生物制药价值的人类蛋白质来验证该工作流程，这些蛋白质要么是直接治疗药物，要么是研究靶标，它们的大小和 GC 含量连续分布。这包括证明该工作流程适用于单克隆抗体及其单链抗体片段衍生物的三部分组装。该工作流程应该能够促进未来对毕赤酵母重组蛋白生产的研究和对该工业宿主的合成生物学方法的研究。
