High-throughput insect cell protein expression applications.
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.
Buchs
Mirjam
M
Biologics Center, Novartis Institutes for Biomedical Research, Basel, Switzerland.
Kim
Ernie
E
Pouliquen
Yann
Y
Sachs
Michael
M
Geisse
Sabine
S
Mahnke
Marion
M
Hunt
Ian
I
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000254:growth & development; D018929:Cell Culture Techniques; D002477:Cells / Q000378:metabolism; D007313:Insecta / Q000166:cytology / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection / Q000379:methods
D011994:Recombinant Proteins