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. 2016 Jan 28;7:6. doi: 10.3389/fpls.2016.00006

FIGURE 1.

FIGURE 1

Expression of synthetic FLAG multimers in tobacco seeds. (A) Schematic representation of the plant expression cassette for seed-specific expression of intein-based assembled high molecular weight synthetic FLAG. Abbreviations: USP, unknown seed protein promoter; SP, legumin B4 signal peptide; KDEL, ER retention sequence; IntC/IntN, C- and N-terminal intein sequences of the Synechocystis sp. DnaB; GS-linker, flexible (GGGGS)3 spacer; c-myc, immunodetection tag. (B) Extracts of T1 seeds of four different transformed tobacco plants expressing the FLAG-Intein-c-myc protein (USP-FIC) and of corresponding wild type cultivar Nicotiana tabacum cv. Samsun NN were separated by gradient SDS-PAGE (4–10% PAA) with 30 μg total soluble seed protein loaded per lane reflecting 4.1 seeds (USP-FIC 28), 4.0 seeds (USP-FIC 14), 4.7 seeds (USP-FIC 10), and 3.9 seeds (USP-FIC 5). FLAG multimers were immunodetected by Western blotting based on the c-myc tag. c-myc standards (a) 0.5 ng, (b) 1 ng, (c) 2 ng of anti-TNF-VHH-100xELP (Conrad et al., 2011); WT, wild type.