Co-expression of a scFv antibody fragment and a reporter protein using lentiviral shuttle plasmid containing a self-processing furin-2A sequence

Sarah L. Appleby, Yazad Irani, Lauren A. Mortimer, Helen M. Brereton, Sonja Klebe, Miriam C. Keane, Peter J. Cowan, Keryn A. Williams

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

It is often desirable to co-express a reporter protein with a potential therapeutic protein, to verify correct targeting of an expression strategy. Vectors containing a viral self-processing 2A sequence have been reported to drive equimolar expression of two or more transgenes from a single promoter. Here, we report on the co-expression of a secreted antibody fragment and an intracellular reporter protein, enhanced yellow fluorescent protein from lentiviral shuttle plasmids by inserting a furin-2A (F2A) sequence between the two cDNAs, in two different orientations, in the expression cassette. We show that the order of these two transgenes relative to the F2A sequence affects expression levels. Reduced expression of each transgene positioned downstream of F2A, compared with upstream of F2A, was observed (p. <. 0.05). Moreover, protein expression from double-cDNA plasmids was significantly lower than from their corresponding single transgene counterparts (p. <. 0.05).

LanguageEnglish
Pages61-65
Number of pages5
JournalJournal of Immunological Methods
Volume397
Issue number1-2
DOIs
Publication statusPublished - 2013

Keywords

  • 2A self-processing sequence
  • Co-expression
  • Furin cleavage site
  • Lentiviral vector
  • Reporter protein
  • ScFv

ASJC Scopus subject areas

  • Immunology
  • Immunology and Allergy

Cite this

Appleby, Sarah L. ; Irani, Yazad ; Mortimer, Lauren A. ; Brereton, Helen M. ; Klebe, Sonja ; Keane, Miriam C. ; Cowan, Peter J. ; Williams, Keryn A. / Co-expression of a scFv antibody fragment and a reporter protein using lentiviral shuttle plasmid containing a self-processing furin-2A sequence. In: Journal of Immunological Methods. 2013 ; Vol. 397, No. 1-2. pp. 61-65.
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Co-expression of a scFv antibody fragment and a reporter protein using lentiviral shuttle plasmid containing a self-processing furin-2A sequence. / Appleby, Sarah L.; Irani, Yazad; Mortimer, Lauren A.; Brereton, Helen M.; Klebe, Sonja; Keane, Miriam C.; Cowan, Peter J.; Williams, Keryn A.

In: Journal of Immunological Methods, Vol. 397, No. 1-2, 2013, p. 61-65.

Research output: Contribution to journalArticle

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AU - Mortimer, Lauren A.

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AU - Klebe, Sonja

AU - Keane, Miriam C.

AU - Cowan, Peter J.

AU - Williams, Keryn A.

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