Substrate limitation in the baculovirus expression vector system

✍ Scribed by Kathryn M. Radford; Steven Reid; Paul F. Greenfield

• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: English
• Weight: 411 KB
• Volume: 56
• Category: Article
• ISSN: 0006-3592
• DOI: 10.1002/(sici)1097-0290(19971005)56:1<32::aid-bit4>3.0.co;2-w

✦ Synopsis

The inability to infect insect cell cultures at the highest achievable cell densities has imposed major limitations to both the fundamental understanding of the Baculovirus Expression Vector System (BEVS) as well as full exploitation of its potential productive capacity for recombinant (␤-galAcNPV) products. The current literature does not characterize and identify the exact nature of the observed limitations, which therefore has become the major objective and contribution of the following study. Critical densities for infection of Spodoptera frugiperda (Sf9) cells with nuclear polyhedrosis virus ex- pressing ␤-galactosidase (Autographa californica) grown in media both containing fetal calf serum (FCS) and free of serum were found to be at 2 × 10 6 and 5 × 10 6 cells/ml respectively. Medium exchange was found to completely reverse the effect if renewed up to 24 hours postinfection (HPI). The inevitable arrest of uninfected cell growth and decreased production of recombinant products at high cell densities of infection were both correlated to nutrient depletion. Cystine was found to be depleted in uninfected insect cell cultures at the onset of the stationary phase and in serum-free insect cell cultures infected with baculovirus above a cell density of 5 × 10 6 cells/ml. Neither glucose depletion nor accumulation of possible inhibitory metabolites such as alanine, ammonia, or lactate could be correlated to growth arrest or decreased recombinant product yields.