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

João Miguel Nunes Vidigal, Bárbara Fernandes, Mafalda M. Dias, Marco Patrone, António Roldão, Manuel J.T. Carrondo, P.M. Alves, Ana Palma Teixeira

Research output: Contribution to journalArticle

11 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)655-666
Number of pages12
JournalApplied Microbiology and Biotechnology
Volume102
Issue number2
DOIs
Publication statusPublished - 1 Jan 2018

Keywords

  • GPCRs
  • Insect cell line development
  • Membrane proteins
  • RMCE systems
  • Virus-like particles (VLPs)

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