RMCE-based insect cell platform to produce membrane proteins captured on HIV-1 Gag virus-like particles

Abstract

Conformationally complex membrane proteins (MPs) are 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 is a promising approach to obtain correctly folded proteins at the surface of virus-like particles (VLPs), preserving their native lipidic environment. Here, we implemented a site-specific recombinase-mediated cassette exchange (RMCE) strategy to establish a reusable HIV-1 Gag-expressing insect cell line for fast production of target MPs on the surface of Gag-VLPs. The Sf9 cell line was initially tagged with a Gag-GFP-expressing cassette incorporating two flipase recognition target sites (FRTs), one within the fusion linker of Gag-GFP. The GFP cassette was afterwards replaced by a Cherry cassette via flipase (Flp) recombination. The fusion of Gag to fluorescent proteins enabled high-throughput screening of cells with higher Gag expression and Flp-mediated cassette exchange ability, while keeping the functionality of the VLP scaffold unaltered. The best cell clone was then Flp-recombinated to produce Gag-VLPs decorated with a human β2-adrenergic receptor (β2AR). Release of a fluorescently labeled β2AR into the culture supernatant was confirmed by immunoblotting, and its co-localization with Gag-VLPs was visualized by confocal microscopy. Furthermore, the differential avidity of β2AR-dsplaying Gag-VLPs versus "naked" Gag-VLPs to an anti-β2AR antibody measured by ELISA corroborated the presence of β2AR at the surface of the Gag-VLPs. In conclusion, this novel insect cell line represents a valuable platform for fast production of MPs in their native conformation, which can accelerate small-molecule and antibody drug discovery programs.

Single-cell cloning enables the selection of more productive Drosophila melanogaster S2 cells for recombinant protein expression

“…Consistent with the statements above, our polyclonal cell lines showed variable cell-specific expression levels. This is in agreement with previous reports concerning other polyclonal rS2 cells expressing EGFP [ 37 ] as well as polyclonal Sf9 cells expressing GFP-tagged virus-like particles [ 38 ]. To overcome the limitations of polyclonality, we isolated monoclonal lines by limiting dilution, which not only unified the expression profile but also achieved a multifold increase in cell-specific productivity.…”

“…After separation by FACS, single rS2 cells are expanded using essentially the same protocol as for limiting dilution [ 48 ]. FACS is useful, especially in the context of cell line generation by targeted gene insertion using recombinase mediated cassette exchange (RMCE), because here the fluorescent tagging cassette is later exchanged for a targeting cassette and consequently there is a direct relationship between the preliminary fluorescence signal and the final product expression level [ 38 , 48 , 49 ]. However, unless the reporter is exchanged by RMCE or directly fused to the target protein, the use of fluorescence for selection is problematic.…”

Accelerating HIV‐1 VLP production using stable High Five insect cell pools

“…[27] High Five cells were transfected with the cationic lipid reagent Cellfectin II (Thermo Fisher Scientific) as previously reported. [18] Briefly, 0.3 µg 10 −6 cells of linearized pIZTV5-mCherry or pIZTV5-Gag-eGFP plasmid and 0.8 µg 10 −6 cells of Cellfectin II were separately added to 500 µL of non-supplemented Grace's insect medium (Thermo Fisher Scientific), vortexed for 5 s, and added to the culture. Digestion with…”

“…[16,17] As for VLPs, insect cells are a highly productive system with successful results reported by SGE using clonal cell lines. [18,19] However, alternative stable production strategies allowing bioprocess acceleration remain to be investigated. The transfectability [20,21] and capacity of High Five cells to produce large VLP yields [22,23] make them an interesting system for this purpose.…”

Coupling Microscopy and Flow Cytometry for a Comprehensive Characterization of Nanoparticle Production in Insect Cells

“…virus (HIV) is conventionally the preferred scaffold for protein display in the lipid envelope (5,6).…”