Abstract
Here we describe the development of a baculovirus vector expression cassette containing rearranged baculovirus-derived genetic regulatory elements. This newly designed expression cassette conferred significant production improvements to the baculovirus expression vector system (BEVS), including prolonged cell integrity after infection, improved protein integrity, and around 4-fold increase in recombinant protein production yields in insect cells with respect to a standard baculovirus vector. The expression cassette consisted of a cDNA encoding for the baculovirus transactivation factors IE1 and IE0, expressed under the control of the polyhedrin promoter, and a homologous repeated transcription enhancer sequence operatively cis-linked to the p10 promoter or to chimeric promoters containing p10. The prolonged cell integrity observed in cells infected by baculoviruses harbouring the novel expression cassette reduced the characteristic proteolysis and aberrant forms frequently found in baculovirus-derived recombinant proteins. The new expression cassette developed here has the potential to significantly improve the productivity of the BEVS.
Highlights
The baculovirus expression vector system (BEVS) is one of the most powerful, robust, and versatile eukaryotic expression systems available
The baculovirus vector most commonly used in industry and research laboratories for recombinant protein production is based on Autographa californica multinuclear polyhedrosis virus (AcMNPV) with Spodoptera frugiperda 9 (Sf9) or 21 (Sf21) insect cells, Trichoplusia ni (T. ni)-derived High Five (Hi-5TM) cells, and whole T. ni insect larvae as suitable expression hosts
Like any other expression system, the BEVS has a number of bottlenecks, one of which regards the expression yields obtained in insect cells, which are significantly lower than those achieved with the most productive mammalian cells
Summary
The baculovirus expression vector system (BEVS) is one of the most powerful, robust, and versatile eukaryotic expression systems available. While common expression yields in optimised transformed fed-batch mammalian cells cultured in a bioreactor may reach grams of recombinant protein per L, in insect cells infected by recombinant baculoviruses the yield rarely exceeds 50 to 100 mg per L. This relatively low expression capacity can be compensated in the BEVS by the short development times and lower costs associated with a specific product. A marked proteolysis of recombinant proteins during baculovirus-based production is frequently encountered This observation is due, in part, to the cytopathogenic effects of the baculovirus vectors in insect cells during infection [7,8,9]
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