Using cell size kinetics to determine optimal harvest time for Spodoptera frugiperda and Trichoplusia ni BTI-TN-5B1-4 cells infected with a baculovirus expression vector system expressing enhanced green fluorescent protein
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Infecting insect cells with a baculovirus expression vector system (BEVS) is an increasingly popular method for the production of recombinant proteins. Due to the lytic nature of the system, however, determining the optimal harvest time is critical for maximizing protein yield. We found that measuring the change in average diameter during the progress of infection with an automated cell analysis system (Cedex HiRes, Innovatis AG) could be used to determine the time of maximum protein production and, thus, optimal harvest time. As a model system, we use insect cells infected with a baculovirus expressing enhanced green fluorescent protein (EGFP). We infected two commonly used insect cell lines, Spodoptera frugiperda (Sf-9) and Trichoplusia ni BTI-TN-5B1-4 (Hi5) with an Autographa californica nuclear polyhedrosis virus (AcNPV) encoding EGFP at various multiplicities of infection (MOI). We monitored the progress of infection with regard to viability, viable cell density and change in average cell diameter with a Cedex HiRes analyzer and compared the results to the EGFP produced. Peak protein production was reached one to two days after the point of maximum average diameter in all conditions. Thus, optimal harvest time could be determined by monitoring the change in average cell diameter during the course of an infection of a cell culture.
KeywordsBaculovirus BEVS Sf-9 Hi5 Cell size kinetics Average cell diameter EGFP Cedex HiRes
baculovirus expression vector system
Trichoplusia ni BTI-TN-5B1-4
Autographa californica nuclear polyhedrosis virus
enhanced green fluorescent protein
multiplicity of infection
plaque forming units
relative fluorescence intensity
The authors would like to thank Per-Erik Strömstedt and Philippe Cronet for their unfailing support of this project; Catherine Heddle for very kindly providing the recombinant EGFP-AcNPV baculovirus; and Thomas Becker for critically reading the manuscript.
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