SARS-CoV-2 (D614) Spike Expression Vectors

Original Spike (D614) Expression vectors

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). 

More detailsLearn 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)

More details More details

 

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.

Wuhan-Hu-1 Spike (D614) Expression vectors
Wuhan-Hu-1 Spike (D614) Expression vectors

SPECIFICATIONS

Specifications

Gene
Optimized SARS-CoV-2 Spike gene (D614)
Accession number

NC_045512.2

ORF size
3765 bp
Plasmid backbone
pUNO
Antibiotic resistance
Blasticidin
Gene promoter
hCMV (human cytomegalovirus) enhancer & promoter
Subclone start
AgeI
Subclone end
NheI
Purification
Ion-exchange chromatography
Reconstitution buffer
Sterile water
Tested applications

Cell fusion assays, flow cytometry

Quality control

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

  • Product: 
    pUNO1-Spike
  • Cat code: 
    p1-spike
  • Quantity: 
    20 µg
Includes:

2 x 1 ml of Blasticidin (10 mg/ml)

Shipping & Storage

  • Shipping method:  Room temperature

    • Storage:

    • -20°C
    Stability: Resuspended DNA should be stored at -20°C and is stable for at least 1 year.

    Caution:

    • Avoid repeated freeze-thaw cycles

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

pUNO1-Spike

Technical Data Sheet

Plasmid Sequence

Safety Data Sheet

Plasmid Map and Sequence

Certificate of analysis

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