Graduate Program in Cell, Molecular, and Structural Biology, Miami University, Oxford, OH, 45056 USA; Department of Microbiology, Miami University, Oxford, OH, 45056 USA.
Department of Microbiology, Miami University, Oxford, OH, 45056 USA.
J Biotechnol. 2017 Aug 10;255:37-46. doi: 10.1016/j.jbiotec.2017.06.397. Epub 2017 Jun 20.
The Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-based Bac-to-Bac expression system consists of a bacmid and five pFastBac™ donor transfer vectors. It has been widely used for eukaryotic gene expression in insect cells to elucidate gene function in biotechnology laboratories. The pFastBac™ vectors contain a 50bp AcMNPV polyhedrin (polh) promoter and a 127bp SV40 polyadenylation (pA) signal for cloning a gene of interest into the bacmid, resulting in unsolved lower gene expression levels than the wild type (wt) AcMNPV in insect cells. Therefore, the purpose of this research is to understand why the Bac-to-Bac system produces lower gene expression levels. Here, we determined that bacmids transposed with pFastBac™ vectors produced 3-4 fold lower levels of certain proteins than the wt AcMNPV. We found that an 80bp cis element 147bp upstream of the 50bp polh promoter and a 134bp polh pA signal are required in pFastBac™ to achieve bacmid protein expression levels equivalent to wt AcMNPV in High Five insect cells. Therefore, researchers currently using pFastBac™ vectors for protein expression can transfer their genes of interest into the improved vectors in this report to elevate protein expression yields in insect cells to reduce protein production costs.
基于 Autographa californica 多角体病毒（AcMNPV）的 Bac-to-Bac 表达系统由一个 bacmid 和五个 pFastBac™ 供体质粒组成。它已被广泛用于昆虫细胞中的真核基因表达，以阐明生物技术实验室中的基因功能。pFastBac™ 载体包含一个 50bp AcMNPV 多角体蛋白（polh）启动子和一个 127bp SV40 多聚腺苷酸化（pA）信号，用于将感兴趣的基因克隆到 bacmid 中，导致昆虫细胞中的基因表达水平低于野生型（wt） AcMNPV。因此，本研究的目的是了解 Bac-to-Bac 系统为何产生较低的基因表达水平。在这里，我们确定用 pFastBac™ 载体转座的 bacmid 产生的某些蛋白质水平比 wt AcMNPV 低 3-4 倍。我们发现，在 50bp polh 启动子上游 147bp 的一个 80bp 顺式元件和一个 134bp polh pA 信号对于 pFastBac™ 是必需的，以达到在 High Five 昆虫细胞中与 wt AcMNPV 相当的 bacmid 蛋白表达水平。因此，目前使用 pFastBac™ 载体进行蛋白质表达的研究人员可以将他们感兴趣的基因转移到本报告中改进的载体中，以提高昆虫细胞中的蛋白质表达产量，从而降低蛋白质生产成本。
