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

J Vidigal, B Fernandes, MM Dias, M Patrone, A Roldão, MJT Carrondo, PM Alves, AP Teixeira

Research output: Contribution to journalArticle

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. © 2017 Springer-Verlag GmbH Germany, part of Springer Nature
Original languageEnglish
Pages (from-to)655-666
Number of pages12
JournalApplied Microbiology and Biotechnology
Volume102
Issue number2
DOIs
Publication statusPublished - 2018

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    Vidigal, J., Fernandes, B., Dias, MM., Patrone, M., Roldão, A., Carrondo, MJT., Alves, PM., & Teixeira, AP. (2018). RMCE-based insect cell platform to produce membrane proteins captured on HIV-1 Gag virus-like particles. Applied Microbiology and Biotechnology, 102(2), 655-666. https://doi.org/10.1007/s00253-017-8628-3