pUNO1-Spike
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Cat.code:
p1-spike
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ABOUT
Optimized SARS-CoV-2 Spike gene (D614) for mammalian cell expression
pUNO1-Spike and pUNO1-Spike-dfur plasmids have been specifically designed for the expression of the SARS-CoV-2 Spike (S) protein in mammalian cells. These plasmids encode the full-length Spike sequence from the original isolate identified in Wuhan, and for optimal cellular expression, it is codon-optimized and the C‑terminal ER-retention signal has been removed [1, 2].
Gene Description
These plasmids encode the Spike protein from the original SARS-CoV-2 isolate, first reported in Wuhan in December 2019 [3]. This sequence is characterized by the presence of D614 and is classified as the root of the pandemic in Clade 19A/ Lineage A (Nextstrain/Pango lineage classification).
Learn more about SARS-CoV-2 variants
The Spike protein contains a furin cleavage site that can affect its cellular expression [4, in-house data]. Therefore, depending on your application InvivoGen offers:
- pUNO1-Spike: with a functional furin cleavage site and recommended for Spike/ACE2 cell fusion assays
- pUNO1-Spike-dfur: with an inactive furin (dfur) cleavage site for improved surface expression and detection (flow cytometry)
General Plasmid Description
These plasmids feature a potent mammalian expression cassette composed of the ubiquitous human EF1α-HTLV composite promoter and the SV40 polyadenylation (pAn) signal. The codon-optimized ORF includes the native SARS-CoV-2 Spike signal sequence. The plasmids are selectable with Blasticidin in both E. coli and mammalian cells (transient and stable transfection).
Applications
- Spike-mediated cell fusion assays with pUNO1-Spike
- Cell surface detection by flow cytometry with pUNO1-Spike-dfur
- Screening of SARS-CoV-2 inhibitors including small molecules, monoclonal antibodies, or convalescent plasma
References:
1. Johnson, M.C. et al. 2020. Optimized Pseudotyping Conditions for the SARS-COV-2 Spike Glycoprotein. J Virol 94.
2. Ou, X. et al. 2020. Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat Commun 11, 1620.
3. Zhou, P. et al. 2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-273.
4. Coutard, B. et al. 2020. The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade. Antiviral Res 176, 104742.
All products are for research use only, and not for human or veterinary use.
SPECIFICATIONS
Specifications
NC_045512.2
Cell fusion assays, flow cytometry
Plasmid construct is confirmed by restriction analysis and full‑length open reading frame (ORF) sequencing. After purification by ion-exchange chromatography, predominant supercoiled conformation is verified by electrophoresis.
CONTENTS
Contents
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Product:pUNO1-Spike
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Cat code:p1-spike
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Quantity:20 µg
2 x 1 ml of Blasticidin (10 mg/ml)
Shipping & Storage
- Shipping method: Room temperature
- -20°C
- Avoid repeated freeze-thaw cycles
Storage:
Caution:
Details
Furin cleavage site in the SARS-CoV-2 Spike protein
A furin cleavage sequence (RRxR) is found within a polybasic cleavage site (681-PRRSR/SVA-688) at the boundary between the S1 and S2 domains (S1/S2) of the Spike protein [1]. Furin is enriched in the Golgi apparatus, where it functions to cleave proteins into their 'mature/active forms'. Specifically, it is suggested that cleavage at this site by furin pre-primes the SARS-CoV-2 S protein during its production. This allows further processing by cell surface host proteases (e.g. TMPRSS2) upon binding to ACE2, which ultimately facilitates viral-host membrane fusion [2,3].
► In a mammalian expression system (e.g. HEK293 cells), to maximize the surface expression of the S protein, the furin cleavage site in InvivoGen's pUNO1-Spike-dfur has been inactivated (in-house data). The crucial recognition residues have been mutated (R683A and R685A) ensuring that the S protein is not cleaved by furin.
Furthermore, the S protein possesses cell-cell fusogenic activity and has been shown to trigger large syncytia formation (multi-nucleated cells). Notably, overexpression of an uncleavable S protein (mutated/inactivated furin cleavage site) has been shown to not induce cell-cell fusion, suggesting that cleavage at the multibasic site is a requirement for syncytia formation [3].
► To study cell-cell fusion by the SARS-CoV-2 spike, InvivoGen offers the pUNO1-Spike plasmid.
References:
1. Coutard, B. et al. 2020. The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade. Antiviral Res 176, 104742.
2. Johnson, B.A. et al. 2021. Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis. Nature
3. Papa, G. et al. 2021. Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion. PLoS Pathog 17, e1009246.
DOCUMENTS
Documents
Technical Data Sheet
Plasmid Map and Sequence
Plasmid Sequence
Safety Data Sheet
Certificate of analysis
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