Enhancement and Prolongation of Baculovirus‐Mediated Expression in Mammalian Cells: Focuses on Strategic Infection and Feeding

Abstract

The baculovirus/insect cell system has been widely used for recombinant protein production. Since the finding that baculovirus was able to infect hepatocytes in 1995, various attempts to utilize baculovirus as a gene delivery vehicle into mammalian cells have been reported. In this study, we intended to explore the possibility of utilizing a baculovirus/mammalian cell system as a nonlytic, continuous protein production system. A recombinant baculovirus vector carrying enhanced green fluorescent protein (EGFP) under the control of cytomegalovirus immediate-early (CMV-IE) promoter was constructed. This virus was used to infect four common mammalian cell lines, and HeLa was found to yield the highest expression level. Additions of butyrate and valproic acid both enhanced the expression level, but butyrate exhibited a more profound effect. More importantly, HeLa cells were found to be superinfected by baculovirus, a result not observed in the conventional baculovirus/insect cell system. The effects of multiplicity of infection (MOI) and infection timing were also compared. High MOI up to 800 increased the expression in the short term (4 days), but the relatively higher cell death and lower cell density compromised the overall protein yield thereafter. The highest overall expression for a long term was obtained at MOI = 200 when the cells were initially infected at the mid-exponential phase and superinfected with additional baculovirus (MOI = 200) together with a one-time supplement of butyrate. In summary, the strategic infection and feeding enhanced the expression level 9-fold (compared with unsuperinfected culture) and prolonged the duration of expression to 16 days. This study reveals that this baculovirus/mammalian cell system has great potential to become a novel continuous, nonlytic expression system.

Generation of iPS Cells Using a BacMam Multigene Expression System

“…It has been reported that the addition of baculovirus to mammalian cells in mid-log phase, followed by three further successive additions of virus (superinfection) at intervals of 3-4 days allows recombinant gene expression to be prolonged for over 10-15 days without addition any enhancing chemical such as butyrate (Hu et al, 2003). However, in our present transduction conditions with the multigene-BacMam vector carrying the VSV-G and WPRE elements, repetitive transduction was not always needed for efficient generation of iPS cells (i.e.…”

A Self-Excisable Infectious Bacterial Artificial Chromosome Clone of Varicella-Zoster Virus Allows Analysis of the Essential Tegument Protein Encoded by ORF9

“…Baculoviruses have been shown to be an effective vehicle for the introduction of DNA sequences into mammalian cells since they neither replicate nor cause cytopathic effects (24). MeWo cells were transfected with pP-Oka9⌬M DNA and infected at 1, 2, 3, 4, or 5 days posttransfection with a recombinant baculovirus expressing ORF9 under the control of the HCMV-IE promoter.…”

Investigation of optimal transduction conditions for baculovirus‐mediated gene delivery into mammalian cells

“…The disadvantage of baculovirus is the transient nature of expression, which usually lasts for 7 days, then the expression declines rapidly. Nonetheless, the expression level can be enhanced and prolonged by superinfection (Hu et al, 2003b).…”