Laboratory of Virology, Wageningen University, Droevendaalsesteeg 1, 6708PB Wageningen, The Netherlands.
Protein Expression Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702, USA.
Viruses. 2020 Dec 16;12(12):1448. doi: 10.3390/v12121448.
Baculovirus expression vectors are successfully used for the commercial production of complex (glyco)proteins in eukaryotic cells. The genome engineering of single-copy baculovirus infectious clones (bacmids) in has been valuable in the study of baculovirus biology, but bacmids are not yet widely applied as expression vectors. An important limitation of first-generation bacmids for large-scale protein production is the rapid loss of gene of interest (GOI) expression. The instability is caused by the mini-F replicon in the bacmid backbone, which is non-essential for baculovirus replication in insect cells, and carries the adjacent GOI in between attTn7 transposition sites. In this paper, we test the hypothesis that relocation of the attTn7 transgene insertion site away from the mini-F replicon prevents deletion of the GOI, thereby resulting in higher and prolonged recombinant protein expression levels. We applied lambda red genome engineering combined with SacB counterselection to generate a series of bacmids with relocated attTn7 sites and tested their performance by comparing the relative expression levels of different GOIs. We conclude that GOI expression from the () locus results in higher overall expression levels and is more stable over serial passages compared to the original bacmid. Finally, we evaluated this improved next-generation bacmid during a bioreactor scale-up of Sf9 insect cells in suspension to produce enveloped chikungunya virus-like particles as a model vaccine.
杆状病毒表达载体被成功用于真核细胞中复杂（糖）蛋白的商业生产。 在 中对单拷贝杆状病毒感染性克隆（bacmid）的基因组工程在杆状病毒生物学研究中非常有价值，但 bacmid 尚未广泛用作表达载体。 第一代 bacmid 用于大规模蛋白质生产的一个重要限制是感兴趣基因（GOI）表达的快速丢失。 不稳定性是由 bacmid 骨架中的 mini-F 复制子引起的，该复制子对于杆状病毒在昆虫细胞中的复制不是必需的，并且在 attTn7 转座子之间携带相邻的 GOI。 在本文中，我们检验了这样一个假设，即转座基因插入位点远离 mini-F 复制子的重新定位可以防止 GOI 的缺失，从而导致更高和更持久的重组蛋白表达水平。 我们应用了 lambda red 基因组工程，并结合了 SacB 反向选择，生成了一系列具有重新定位的 attTn7 位点的 bacmid，并通过比较不同 GOI 的相对表达水平来测试它们的性能。 我们得出结论，与原始 bacmid 相比，从 （） 位点表达 GOI 可导致更高的总体表达水平，并且在连续传代过程中更稳定。 最后，我们在 Sf9 昆虫细胞悬浮液的生物反应器放大过程中评估了这种改进的下一代 bacmid，以生产包膜的基孔肯雅病毒样颗粒作为模型疫苗。
