High-Throughput Insect Cell Protein Expression Applications

Abstract

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.

Recent advances in the production of proteins in insect and mammalian cells for structural biology

“…Under these conditions cells can be grown to a density of $5 Â 10 6 cells/ml of culture, which is adequate for expression studies. Interestingly, it has been reported that P 1 viral titres obtained from amplification of viruses in suspension are usually 10-fold higher ($5 Â 10 8 pfu/ml) than for static cultures (Buchs et al, 2009), removing the need for further rounds of amplification to obtain a high titre viral stock. Although multiplicity of infection can be determined empirically by varying the volume ratio of P 1 virus to the number of cells infected, determining the precise titre of a baculovirus may sometimes be necessary, e.g., trouble-shooting lack of expression.…”

Transient transfection coupled to baculovirus infection for rapid protein expression screening in insect cells

“…Recently published high-throughput insect cell expression systems have streamlined expression screening and protein analysis, but still adhere to generating and amplifying recombinant baculoviruses for each individual construct (Mancia and Love, 2011;Hanson et al, 2007;Buchs et al, 2009). In Fig.…”

High throughput platforms for structural genomics of integral membrane proteins

“…!,49]. System-based on the infection of insect cells with recombinant baculovirues often produce more proteins per litre, more economically, than mammalian cell expression systems.…”