title: Co-expression of a scFv antibody fragment and a reporter protein using lentiviral shuttle plasmid containing a self-processing furin-2A sequence.
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).
authors.affiliation: Department of Ophthalmology, Flinders University, Adelaide, SA 5042, Australia.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Appleby
authors.forename: Yazad
authors.initials: Y
authors.lastname: Irani
authors.forename: Lauren A
authors.initials: LA
authors.lastname: Mortimer
authors.forename: Helen M
authors.initials: HM
authors.lastname: Brereton
authors.forename: Sonja
authors.initials: S
authors.lastname: Klebe
authors.forename: Miriam C
authors.initials: MC
authors.lastname: Keane
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Cowan
authors.forename: Keryn A
authors.initials: KA
authors.lastname: Williams
mesh_terms: D045683:Furin / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D057127:Single-Chain Antibodies / Q000235:genetics*
chemical_list: D008164:Luminescent Proteins; D057127:Single-Chain Antibodies; D045683:Furin
keywords: 2A self-processing sequence; Co-expression; F2A; Furin cleavage site; GFP; HEK-293A; HSD; Lentiviral vector; MOI; Reporter protein; eYFP; enhanced yellow fluorescent protein; furin-2A composite sequence; green fluorescent protein; honestly significant difference test; human embryonic kidney-293A cell line; multiplicity of infection; scFv; single chain antibody fragment