Fig. 4

Accumulation of DARPin G3 in N. tabacum chloroplasts and purification by the (HE)₃-tag. a Western blot analysis of chloroplast-made DARPin G3 expression in tobacco transplastomic plants. Blots were detected using rabbit anti-His-tag antibody as a primary antibody and goat anti-rabbit conjugated with HRP antibody as a secondary antibody. M: prestained molecular mass markers (kDa); cTSP: Total soluble protein from transplastomic plant leaves; chTSP: total soluble protein from isolated chloroplasts of the transplastomic plants; WT: total soluble protein from wild-type plant. Sizes are shown on the left-hand side in kilodaltons (kDa). b ELISA analysis for the DARPin G3 content expressed in tobacco chloroplast. Wild-type tobacco protein extracted by the same procedure was used as a negative control. A purified 15 kDa His-tagged protein was used as a positive standard. Rabbit anti-His-tag antibody (1:2000) and goat anti-rabbit antibody (1:10,000) were used as primary and secondary antibodies, respectively. Detection was performed with the TMB substrate for 30 min at RT. The measurement was performed in biological triplicates. c Apparent morphological equivalency between wild-type (left) and transplastomic (right) tobacco plants. d SDS–PAGE analysis of purified chloroplast-made DARPin G3 from the total soluble protein of isolated intact chloroplast from transplastomic plants. 12% Tris–Glycine gel stained with Coomassie blue; samples were processed with 2 × SDS–PAGE reducing buffer. M: unstained protein MW marker (Thermo scientific); FT: flow-through from Ni⁺ NTA resin; W1, W2: wash steps; E1–E3: three eluents from IMAC. Purified chloroplast-made DARPin G3 is marked on the gel by an arrow