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

7 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

Fingerprint

Recombinases
Virion
Insects
HIV-1
Membrane Proteins
Adrenergic Receptors
Drug Discovery
Cell Line
Sf9 Cells
Adrenergic Antagonists
Antibodies
Immunoblotting
Confocal Microscopy
Genetic Recombination
Proteins
Clone Cells
Enzyme-Linked Immunosorbent Assay
Viruses

Keywords

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

Cite this

Vidigal, João Miguel Nunes ; Fernandes, Bárbara ; Dias, Mafalda M. ; Patrone, Marco ; Roldão, António ; Carrondo, Manuel J.T. ; Alves, P.M. ; Teixeira, Ana Palma. / RMCE-based insect cell platform to produce membrane proteins captured on HIV-1 Gag virus-like particles. In: Applied Microbiology and Biotechnology. 2018 ; Vol. 102, No. 2. pp. 655-666.
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RMCE-based insect cell platform to produce membrane proteins captured on HIV-1 Gag virus-like particles. / Vidigal, João Miguel Nunes; Fernandes, Bárbara; Dias, Mafalda M.; Patrone, Marco; Roldão, António; Carrondo, Manuel J.T.; Alves, P.M.; Teixeira, Ana Palma.

In: Applied Microbiology and Biotechnology, Vol. 102, No. 2, 01.01.2018, p. 655-666.

Research output: Contribution to journalArticle

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AU - Vidigal, João Miguel Nunes

AU - Fernandes, Bárbara

AU - Dias, Mafalda M.

AU - Patrone, Marco

AU - Roldão, António

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