Protein expression in the baculovirus system.
Insect cell-recombinant baculovirus co-cultures offer a protein production system that complements microbial systems by providing recombinant proteins in soluble form and with most post-translational modifications. Moreover, the large size of the viral genome enables cloning of large segments of DNA and consequent expression of complex protein aggregates. This unit describes methods associated with the large-scale production of recombinant proteins in the baculovirus expression system. A method for large-scale production of viral stocks is described and methods for titration of virus are provided (a plaque assay and an end-point assay). Once viral stocks have been prepared and titered, a protocol for testing the virus in small-scale cultures is provided to determine the kinetics of expression, which allows evaluation of various cell culture and infection conditions aimed at developing optimal levels of protein production (e.g., comparisons of different host cell lines, media, and environmental parameters). Support protocols provide instructions for preparing culture samples for protein analysis by SDS-PAGE and discuss analytical methods for monitoring nutrient levels in cell culture fluids. Once optimal process parameters are identified, protocols describe production of the target protein on a large scale in fermentors using either regular batch production in bioreactors or a fed-batch procedure of production in perfusion cultures. Techniques for harvesting cultures from bioreactors are also provided.
Bernard
A
A
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Payton
M
M
Radford
K R
KR
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000254:growth & development; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera
D011994:Recombinant Proteins