Philipps Björn, Rotmann Daniel, Wicki Micha, Mayr Lorenz M, Forstner Michael
Discovery Technologies, Novartis Institutes for BioMedical Research, Basel, Switzerland.
Protein Expr Purif. 2005 Jul;42(1):211-8. doi: 10.1016/j.pep.2005.03.020. Epub 2005 Apr 9.
Rapid expression of recombinant proteins for structure determination is one of the major challenges in pharmaceutical and academic research, since the number of potential drug targets has increased significantly in the last decade. Despite the fact that the baculovirus expression vector system is widely used for this purpose, the system is hampered by three very slow and tedious procedures, namely generation of high titer baculovirus stock, determination of the virus titer and discovery of the best conditions for protein expression. We herein describe the development of the ultraBac system to address and overcome these issues for protein expression in insect cells. We have established a new baculovirus expression technology for insect cells that is based on co-expression of GFP with target genes, a new regime for cell culturing and a highly efficient purification and enrichment procedure for recombinant baculovirus particles. Co-expression of GFP is used to monitor the infection of insect cells, to simplify titer determination and to optimize expression conditions. The new regime for cell culturing with increased viability of non-infected insect cells and its combination with the massive enrichment of virus particles via high-speed centrifugation enables the production of large amounts of recombinant virus in a very short period of time. By combining these techniques and by using the bicistronic vector pUltraBac-1, we have been able to cut the time-lines for protein expression in insect cells by half, approaching those for protein production in Escherichia coli. This new expression system is a significant step forward towards industrialized protein production in both, industry and academia.
用于结构测定的重组蛋白快速表达是制药和学术研究中的主要挑战之一，因为在过去十年中潜在药物靶点的数量显著增加。尽管杆状病毒表达载体系统广泛用于此目的，但该系统受到三个非常缓慢且繁琐的程序的阻碍，即高滴度杆状病毒储备的产生、病毒滴度的测定以及蛋白质表达最佳条件的发现。我们在此描述了ultraBac系统的开发，以解决和克服昆虫细胞中蛋白质表达的这些问题。我们已经建立了一种用于昆虫细胞的新型杆状病毒表达技术，该技术基于绿色荧光蛋白（GFP）与靶基因的共表达、一种新的细胞培养方案以及一种用于重组杆状病毒颗粒的高效纯化和富集程序。GFP的共表达用于监测昆虫细胞的感染、简化滴度测定并优化表达条件。具有更高未感染昆虫细胞活力的新细胞培养方案及其与通过高速离心大量富集病毒颗粒相结合，能够在非常短的时间内产生大量重组病毒。通过结合这些技术并使用双顺反子载体pUltraBac-1，我们已经能够将昆虫细胞中蛋白质表达的时间缩短一半，接近大肠杆菌中蛋白质生产的时间。这种新的表达系统是朝着工业和学术界工业化蛋白质生产迈出的重要一步。
