Details

Autor(en) / Beteiligte

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

Titel

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

Ist Teil von

• Journal of immunological methods, 2013-11, Vol.397 (1-2), p.61-65

Ort / Verlag

Netherlands: Elsevier B.V

Erscheinungsjahr

2013

Quelle

MEDLINE

Beschreibungen/Notizen

• 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). •Co-expression of scFv protein and eYFP from single SV40 promoter•Genes were linked by furin cleavage site and viral 2A self-processing sequence.•Unequal expression of proteins targeted to different cellular compartments•Single gene plasmids produced higher level of expression.