Significant productivity improvement of the baculovirus expression vector system by engineering a novel expression cassette

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.

Figure 1. Schematic representation of genetic components of the baculovirus expression cassette (TB) developed.

Figure 2. Increase of viability and integrity observed on insect cells infected with a baculovirus vector expressing the cDNA Ac-ie-01, encoding for the transactivators IE1/IE0, under the control of the polh promoter.

A) Viability of cells infected (m.o.i. of 0.1) with control and Ac-ie-01 recombinant baculovirus determined by Trypan blue staining at various times post-infection. B) Micrographs of Hi-5 cells cultured in monolayer and infected by the control or the above mentioned baculovirus at 96 hpi.

Figure 3. Recombinant GFP expression in Sf21 insect cells infected at a m.o.i. of 5 with rBacs carrying different expression regulatory genetic elements.

A) Fluorimetric analysis expressed as arbitrary fluorescence units of cells infected at 96 hpi with the baculoviruses. Bars represent the media and standard deviation of three independent experiments. B) Visual fluorescence of Sf21 or Sf9 insect cells cultured in monolayer or suspension respectively and infected with the indicated baculoviruses at a m.o.i. of 5 (monolayer) or 0.1 (suspension) and observed under UV light.

Figure 4. Production of GFP recombinant protein by insect cells infected by recombinant baculoviruses expressing the protein under the control of the TB1 cassette or the polh promoter, as determined by capillary electrophoresis using the Experion system.

A) Comparison of GFP productivity (percentage of total soluble protein from cell extracts) in Sf9 cells cultured in monolayer and infected at a m.o.i. of 5 with a baculovirus expressing the reporter protein under the control of TB1 expression cassette (black) or under the polh promoter (white). B) Comparison of GFP productivity (percentage of total soluble protein from cell extracts) in Sf21 cells cultured in suspension and infected at a m.o.i. of 0.1 with a baculovirus expressing the reporter protein under the control of the TB3 expression cassette (black) or the polh promoter (white). Coomassie blue staining of SDS-PAGE gels resolving the proteins in the cell extracts in both experimental conditions are also shown in panels A and B. In both cases, the GFP band in cells infected by the baculovirus carrying the TB cassettes was more abundant than that expressed by the conventional baculovirus, thereby evidencing higher production yields and confirming the quantification results obtained by other methods.

Figure 5. Influence of genetic regulatory elements in a TB expression cassette on the conformation, proteolysis and functionality of recombinant proteins expressed by a baculovirus vector.

A) Analysis by SDS-PAGE and Western blot with an anti-GFP antibody of cell extracts recovered at various times post-infection with a recombinant baculovirus expressing the GFP protein under the control of the polh promoter or the TB3 expression cassette. Different reacting protein bands indicated by arrows can be observed at each time point post-infection with the conventional baculovirus while only a reactive band of the expected molecular weight for GFP is observed in the cell extracts from cells infected with the virus engineered with the TB3 cassette. Actin integrity in the different cell extracts was studied by Western blot using a specific antiserum, showing differences of reactivity among the baculoviruses analysed at late times post-infection. B) Fluorescence activity of cell extracts recovered at different times post-infection with the two analysed baculoviruses and determined by fluorimetry. While the values increase over time in the case of TB3-modified baculovirus, the values peaked at 72 hpi in extracts from cells infected by the conventional baculovirus, decreasing significantly at later times post-infection. C) Analysis by SDS-PAGE and Western blot with anti-His tag and anti-tubulin antibodies simultaneously of cell extracts recovered at different times post-infection with a recombinant baculovirus expressing a single-domain antibody fused to GFP protein (VHHGFP) under the control of polh promoter or under the TB3 expression cassette. A number of degradation bands indicated by arrows were clearly observed in cells infected with the conventional baculovirus expressing the fusion protein after 48 hpi, in contrast to that observed when the fusion protein was expressed under the control of TB3 expression cassette. Tubulin integrity determined by Western blot analysis in the different cell extracts showed differences of reactivity among the baculoviruses analysed at late times post-infection.

Figure 6. Analysis of the compatibility of TB cassettes with commercial site-specific transposition and homologous recombination technologies commonly used to generate recombinant baculoviruses.

A) General strategy followed to obtain the recombinant baculoviruses modified by a TB expression cassette using the Bac-to-Bac and FlashBAC ULTRA commercial systems. Only one additional cloning step is required to introduce the TB expression cassettes into any commercial methodology used to produce a recombinant baculovirus. B) Visual fluorescence of Sf21 insect cells infected at 72 hpi by conventional or TB3-modified baculoviruses under UV illumination. This panel shows the cells both macroscopically and in micrographs obtained by a fluorescence inverted microscope. Fluorescence intensities were highly evident when the baculoviruses expressed the GFP protein under the control of TB3. These differences in fluorescence intensity were not significantly different between the two technologies used to generate the recombinant baculoviruses. C) Quantification by fluorimetry and Coomassie blue staining of SDS-PAGE gels of the GFP in cell extracts at 72 hpi with the different baculoviruses. Highly significant quantitative differences in fluorescence values and protein staining were observed between conventional baculoviruses and those genetically engineered with the TB3 expression cassette.