ena-STUDY-IBET - INSTITUTO DE BIOLOGIA EXPERIMENTAL E TECNOLOGICA-21-09-2017-11:58:30:490-859
RMCE-based insect cell platform to produce membrane proteins captured on HIV-1 Gag virus-like particles
Conformationally-complex membrane proteins (MPs) are major therapeutic targets in many diseases, but drug discovery has been slowed down by the lack of efficient production tools. Co-expression of MPs with matrix proteins from enveloped viruses has been pursued to obtain correctly folded proteins at the surface of virus-like particles (VLP), preserving their native lipidic environment. Here, we implemented a novel recombinase-based strategy to establish an HIV-1 Gag-expressing insect cell line that could be re-used for fast production of Gag-VLPs pseudotyped with target MPs. The Spodoptera frugiperda Sf9 cell line was initially tagged with a Gag-GFP expressing cassette incorporating two flipase recognition target sites (FRTs), one of them within the fusion linker of Gag and GFP. This cassette was afterwards successfully replaced by a Gag-Cherry cassette via Flp-recombination. These two fusion proteins enabled high-throughput fluorescence-activated cell sorting of cells tagged in loci supporting high Gag expression and Flp recombination, while keeping the functionality of the VLP scaffold unaltered. After clone screening, a cell line with significantly high Gag secretion rate and a growth profile similar to that of the parental Sf9 cells was used to produce Gag-VLPs decorated with a human ?2-adrenergic receptor (?2AR), which was integrated in the tagged locus via Flp-recombination. Release of a fluorescently tagged ?2AR into the culture supernatant was confirmed by Western blot and its co-localization with Gag-VLPs was visualized by confocal microscopy in supernatant samples. Furthermore, the differential avidity of the Gag-?2AR VLPs versus “naked” Gag-VLPs to an anti-?2AR antibody measured by ELISA corroborated the presence of ?2AR at the surface of the Gag-VLPs. In conclusion, the insect cell line here developed represents a valuable cell platform for fast production of MPs in their native conformation, which can accelerate small-molecule and antibody drug discovery programs.
Conformationally-complex membrane proteins (MPs) are major therapeutic targets in many diseases, but drug discovery has been slowed down by the lack of efficient production tools. Co-expression of MPs with matrix proteins from enveloped viruses has been pursued to obtain correctly folded proteins at the surface of virus-like particles (VLP), preserving their native lipidic environment. Here, we implemented a novel recombinase-based strategy to establish an HIV-1 Gag-expressing insect cell line that could be re-used for fast production of Gag-VLPs pseudotyped with target MPs. The Spodoptera frugiperda Sf9 cell line was initially tagged with a Gag-GFP expressing cassette incorporating two flipase recognition target sites (FRTs), one of them within the fusion linker of Gag and GFP. This cassette was afterwards successfully replaced by a Gag-Cherry cassette via Flp-recombination. These two fusion proteins enabled high-throughput fluorescence-activated cell sorting of cells tagged in loci supporting high Gag expression and Flp recombination, while keeping the functionality of the VLP scaffold unaltered. After clone screening, a cell line with significantly high Gag secretion rate and a growth profile similar to that of the parental Sf9 cells was used to produce Gag-VLPs decorated with a human ?2-adrenergic receptor (?2AR), which was integrated in the tagged locus via Flp-recombination. Release of a fluorescently tagged ?2AR into the culture supernatant was confirmed by Western blot and its co-localization with Gag-VLPs was visualized by confocal microscopy in supernatant samples. Furthermore, the differential avidity of the Gag-?2AR VLPs versus “naked” Gag-VLPs to an anti-?2AR antibody measured by ELISA corroborated the presence of ?2AR at the surface of the Gag-VLPs. In conclusion, the insect cell line here developed represents a valuable cell platform for fast production of MPs in their native conformation, which can accelerate small-molecule and antibody drug discovery programs.