Expression of recombinant proteins in insect cells by their direct infection with Escherichia coli transformed with baculovirus bacmids

Abstract

Summary. -The baculovirus-insect cell expression system (BES), one of the most popular systems for expression of eukaryotic proteins, was known to have drawbacks such as laborious manipulation of large-size baculovirus bacmids and the transfection procedure. These problems could be eliminated by direct infection of eukaryotic cells with nonpathogenic bacteria harbouring the respective gene -bactofection. However, it was unknown whether this system could be applied to insect cells. Therefore, in this study, the possibility of delivery of enhanced green fluorescent protein (EGFP) gene as a marker into the insect cell lines Sf9 and BmN-SWU1 using the above-mentioned approach with the Bac-to-Bac system was investigated. Using a simple co-incubation of Escherichia coli strains containing recombinant baculovirus bacmids with the EGFP gene and insect cells, it was possible to demonstrate the EGFP expression in these cells and to obtain high-titer recombinant baculoviral stocks. Furthermore, BmN-SWU1 cells proved more susceptible to the used E. coli strains than Sf9 cells. However, the co-expression of invasin and listeriolysin-O, known to enhance the E. coli-mediated gene delivery to mammalian cells, with EGFP, had no effect on insect cells. Summing up, this study proved that a heterologous gene can be efficiently delivered and expressed in insect cells by their simple incubation with non-pathogenic E. coli strains harboring recombinant baculovirus bacmids with the respective gene.Keywords: baculovirus; bacmid; EGFP; Escherichia coli; insect cell * Corresponding authors. E-mail: yxiaolan@163.com, malixin9@ hotmail.com; phone: +86-27-88666349. Abbreviations: AcNPV = Autographa californica nuclear polyhedrosis virus; BES = baculovirus-insect cell expression system; BmNPV = Bombyx mori nuclear polyhedrosis virus; BmN-SWU1 = ovarian cell line of Bombyx mori; EGFP = green fluorescent protein; MOI = multiplicity of infection; p.i. = post infection; sf9 = Spodoptera frugiperda 9; t.o.i. = time of infection

Characterization of an iron-inducible Haemaphysalis longicornis tick-derived promoter in an Ixodes scapularis-derived tick cell line (ISE6)

“…Recombinant proteins are important in the development of tick control strategies especially in vaccine design [22]. Insect cell lines are frequently utilized in recombinant protein production [23]. This strategy could be explored in tick cell lines in the development of expression system for tick-specific recombinant proteins.…”

Protective immune response of recombinant Fiber-2 protein as subunit vaccine against Fowl-adenovirus-4 infection in Pakistan

“…Considering the importance of Fiber-2 proteins and limitations of baculovirus expression such as extensive manipulation to design plasmid, transfection process and use of cell culture [23], E. coli was used to generate the fiber-2 protein, which was then purified using metal affinity chromatography. The recombinant fiber-2 protein was used as subunit vaccine and its efficacy was analyzed by clinical observation and protection exerted following the virus challenge.…”