Buchs Mirjam, Kim Ernie, Pouliquen Yann, Sachs Michael, Geisse Sabine, Mahnke Marion, Hunt Ian
Biologics Center, Novartis Institutes for Biomedical Research, Basel, Switzerland.
Methods Mol Biol. 2009;498:199-227. doi: 10.1007/978-1-59745-196-3_14.
The Baculovirus Expression Vector System (BEVS) is one of the most efficient systems for production of recombinant proteins and consequently its application is wide-spread in industry as well as in academia. Since the early 1970s, when the first stable insect cell lines were established and the infectivity of bacu-lovirus in an in vitro culture system was demonstrated (1, 2), virtually thousands of reports have been published on the successful expression of proteins using this system as well as on method improvement. However, despite its popularity the system is labor intensive and time consuming. Moreover, adaptation of the system to multi-parallel (high-throughput) expression is much more difficult to achieve than with E. coli due to its far more complex nature. However, recent years have seen the development of strategies that have greatly enhanced the stream-lining and speed of baculovirus protein expression for increased throughput via use of automation and miniaturization. This chapter therefore tries to collate these developments in a series of protocols (which are modifications to standard procedure plus several new approaches) that will allow the user to expedite the speed and throughput of baculovirus-mediated protein expression and facilitate true multi-parallel, high-throughput protein expression profiling in insect cells. In addition we also provide a series of optimized protocols for small and large-scale transient insect cell expression that allow for both the rapid analysis of multiple constructs and the concomitant scale-up of those selected for on-going analysis. Since this approach is independent of viral propagation, the timelines for this approach are markedly shorter and offer a significant advantage over standard bacu-lovirus expression approach strategies in the context of HT applications.
杆状病毒表达载体系统（BEVS）是生产重组蛋白最有效的系统之一，因此其应用在工业界和学术界都很广泛。自20世纪70年代初首次建立稳定的昆虫细胞系并证明杆状病毒在体外培养系统中的感染性以来（1,2），实际上已有数千篇关于使用该系统成功表达蛋白质以及方法改进的报道。然而，尽管该系统很受欢迎，但它劳动强度大且耗时。此外，由于其性质更为复杂，与大肠杆菌相比，该系统更难适应多平行（高通量）表达。然而，近年来已经开发出一些策略，通过使用自动化和小型化极大地提高了杆状病毒蛋白表达的流程优化和速度，以提高通量。因此，本章试图在一系列方案（这些方案是对标准程序的修改以及几种新方法）中整理这些进展，这将使用户能够加快杆状病毒介导的蛋白表达的速度和通量，并促进在昆虫细胞中进行真正的多平行、高通量蛋白表达分析。此外，我们还提供了一系列用于小规模和大规模瞬时昆虫细胞表达的优化方案，这些方案既允许快速分析多个构建体，又能对那些被选中进行持续分析的构建体进行相应的放大。由于这种方法不依赖于病毒繁殖，在高通量应用的背景下，这种方法的时间线明显更短，比标准的杆状病毒表达方法策略具有显著优势。
