Characterization of Alternative Promoters to Stagger and Control Protein Expression in the Baculovirus-Insect Cell System: From Intracellular Reporter Proteins to Fluorescent Influenza Virus-like Particles

Abstract

Background The Baculovirus Expression Vector System (BEVS) is increasingly used for protein production in both industry and academia, and much work has been conducted to improve this system. The baculovirus infection of an insect cell sets up a sophisticated and complex series of gene expression events that are very tightly temporally regulated. The study of this system has progressed to such an extent that many control elements, such as activators, enhancers, and promoters involved in this process have been discovered and characterized to some extent, as reviewed in [1]. These control elements can be used to regulate the expression of heterologous genes, in order to move beyond “brute force” expression of large amounts of protein within insect cells. It enables researchers to set up a pre-planned series of expression events of multiple genes within one cell, and to essentially “program” gene expression by modifying the baculovirus genome. While some groups have investigated this, a systematic study of control elements and how expression from a single gene affects expression from other heterologous genes, has not been conducted thus far. This study characterizes gene expression from several baculovirus promoters for the production of proteins and virus-like particles, and examines interaction effects when promoters drive expression of genes at different times and at different levels. Materials and Methods Two sets of protein coding genes were investigated. Both sets of constructs were arranged such that one gene was always under the control of the very strong polyhedron (polh) promoter, while the other gene was under the control of the early ie1, late basic, gp64orvcath, or the very late p10 promoters. The first set of proteins examined consisted of two easily traceable fluorescent proteins requiring minimal post-translational processing: the enhanced green fluorescent protein (eGFP, herein referred to as GFP) and a red fluorescent protein (DsRed2 herein referred to as RFP). The RFP gene was always under the control of the polh promoter while GFP was placed downstream of one of the other five promoters [2].The second set of proteins studied were fusions of influenza A virus proteins. More specifically, human influenza A/PR/8/34 hemagglutinin (HA) and matrix (M1) proteins were fused to eGFP(HAGFP) and DsRed2 (M1RFP) respectively. The M1RFP gene was always under the control of the polh promoter while HAGFP was placed downstream of one of the other five promoters. Sf9 cells were infected at a cell density of 1 × 106 cells/mL and at a multiplicity of infection of 5. Cells were examined by light and fluorescence microscopy, as well as by flow cytometry. Virus-like particles were recovered from infected cell culture supernatants by subjecting the supernatants to iodixanol gradient ultracentrifugation as previously described in [3]. Virus-like particles were characterized by flow cytometry using a method similar to that described in [4],by negative stain * Correspondence: marc.aucoin@uwaterloo.ca Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, N2L3G1, Canada George and Aucoin BMC Proceedings 2015, 9(Suppl 9):P49 http://www.biomedcentral.com/1753-6561/9/S9/P49