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

Sarah Appleby, Yazad Irani, Lauren Mortimer, Helen Brereton, Sonja Klebe, Miriam Keane, Peter Cowan, Keryn 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).

    Original languageEnglish
    Pages (from-to)61-65
    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

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