Time reduction and process optimization of the baculovirus expression system for more efficient recombinant protein production in insect cells
Time reduction and process optimization of the baculovirus expression system for more efficient recombinant protein production in insect cells
复制标题
DOI：
10.1016/j.pep.2005.03.020
复制
发表时间：
2005-07-01
影响因子：
1.6
通讯作者：
Forstner, M
中科院分区：
文献类型：
--
作者：
Philipps, B;Rotmann, D;Forstner, M
快速表达重组蛋白以确定结构是制药和学术研究的主要挑战之一，因为潜在药物靶点的数量在过去十年中显着增加。尽管杆状病毒表达载体系统被广泛用于此目的，但该系统受到三个非常缓慢和繁琐的程序的阻碍，即高滴度杆状病毒原液的产生，病毒滴度的测定和最佳蛋白表达条件的发现。我们在此描述了ultraBac系统的发展，以解决和克服昆虫细胞中蛋白质表达的这些问题。我们建立了一种新的基于GFP与靶基因共表达的昆虫细胞杆状病毒表达技术，一种新的细胞培养制度，以及一种高效的重组杆状病毒颗粒纯化和富集工艺。GFP共表达用于监测昆虫细胞感染，简化滴度测定和优化表达条件。新的细胞培养方法提高了未感染昆虫细胞的生存能力，并通过高速离心使病毒颗粒大量富集，从而能够在很短的时间内产生大量重组病毒。通过结合这些技术和使用双链载体pUltraBac-1，我们已经能够将昆虫细胞中蛋白质表达的时间线缩短一半，接近大肠杆菌中蛋白质生产的时间线。这一新的表达系统是工业和学术界向工业化蛋白质生产迈出的重要一步。(c) 2005爱思唯尔公司版权所有。
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. (c) 2005 Elsevier Inc. All rights reserved.