Plant materials

Tobacco (Nicotiana tabacum Cv. Perega) plants harbor pPRV::DARPin G3 constructs expressing the chloroplast-made DARPin G3 used in this study were obtained from our previous study (Fig. 6) [19]. Briefly, the DARPin G3 expression cassette was constructed by fusing the codon-optimized coding region of DARPin G3 with the strong rRNA operon promoter (Prrn) and a T7g10 5´UTR-derived leader sequence at the 5´-end and the rrnB 3´UTR from E. coli at the 3´-end. The 5´UTR sequence contained a strong ribosome binding site (TAAGGAGTG), an epsilon motif as an enhancer sequence (TTAACTTTA) and a 10-nucleotide poly-A-spacer between the epsilon motif and the ribosome binding site. To facilitate the purification of the protein, the histidine-glutamate (HE)3-tag sequence was embedded at the 5´-end of the DARPin G3 encoding sequence. The final DARPin G3 encoding cassette with translation control elements was subcloned into pPRV111A [52]. The transformation was performed using the particle bombardment method on tobacco leaf explants and the regeneration of transplastomic plants in the selection medium containing 500 mg/l streptomycin [53]. Homoplasmic shoots were regenerated three times under stringent spectinomycin/streptomycin selection pressure. The preliminary confirmation of transgenesis and homoplasmic status of transplastomic plants was confirmed by polymerase chain reaction and Southern blot analysis, respectively. After three rounds of selection, inheritance assays of transplastomic seeds displayed a homogeneous population of antibiotic-resistant seedlings, confirming that they are homoplasmic [54].

Fig. 6

Physical map of fine structure for the chloroplast-specific DARPin G3 expression cassette, Nt-Prrn: ribosomal RNA operon promoter from tobacco; T7g10 5´ UTR: 5´ untranslated region of bacteriophage T7 gene 10; DARPin G3: coding sequence of DARPin G3, TrrnB: rrnB 3´ untranslated region from E. coli; PpsbA: promoter and 5´ UTR of the psbA gene; aadA: aminoglycoside 3´- adenylytransferase gene; TpsbA: terminator of the psbA gene

Isolation of chloroplasts

To prepare chloroplast proteins for downstream analysis, chloroplasts were separated from mature, green, and fully grown leaves from the mid-section of DARPin G3 transplastomic and wild-type tobacco plants, which were kept in the dark for 48 h to destarch the plastids. The leaves of transplastomic and wild-type plants, after removing the midrib, were finely ground and homogenized with 3 volumes (v/w) of ice-cold chloroplast isolation buffer (50 mM Tris-HCl, 0.35 M mannitol, 5 mM disodium EDTA, 0.1% BSA (w/v), and 1.0 mM 2-mercaptoethanol) using a motor-driven blender. The homogenate was centrifuged at 4 °C for 20 min at 1000 × g to pellet cell debris and nuclei after being passed through three layers of Miracloth. To isolate chloroplasts, the supernatant was decanted into fresh tubes and centrifuged at 4 °C for 20 min at 2500 × g. After discarding the supernatant, the green pellet was washed twice in the isolation buffer before final centrifugation for 10 min at 1500 × g. The supernatant was discarded, and the chloroplast pellet was utilized, either to extract total soluble proteins or by lyophilization.

Lyophilization

Fully expanded leaves from transplastomic and wild-type tobacco plants expressing DARPin G3, after excision of the midrib, were sliced into small pieces measuring roughly 1 cm2, frozen in liquid nitrogen, and then lyophilized in a Christ Alpha 1–2 LDplus Freeze Dry System in a vacuum (0.036 mBar) at -55 °C for 72 h. Lyophilization of isolated chloroplasts was performed in vacuum (0.036 mBar) at -50 °C for 72 h. The lyophilized materials were stored at room temperature for nine months and were used for the protein extraction after being ground in a grinder for 2 min at maximum speed three times (each time, pulse on for 15 s and off for 30 s). In the case of lyophilized leaves, the ground materials were subjected to sieving (mesh size: 100).

Protein immunoblot analysis

Total soluble protein was extracted from fresh and lyophilized leaves and chloroplasts of transplastomic and wild-type tobacco plants. 100 mg of fresh, fully expanded leaves grounded in the presence of liquid nitrogen or 100 mg of isolated chloroplasts were combined with 500 µl of cold extraction buffer (PBS 1X, pH 7.4, 150 mM NaCl, and 1X EDTA-free protease inhibitor), vigorously vortexed, and incubated for 30 min at 4 °C. Due to the weight reduction during freeze-drying (93% for leaves and 48% for chloroplast pellets), Lyophilized leaf tissue and chloroplasts were normalized with fresh leaf tissue and fresh isolated chloroplast weight, so that 7 mg of lyophilized leaves and 52 mg of lyophilized chloroplasts were used by adding 500 µl of extraction buffer and vortexed for 1 h at 4 °C for rehydration. The crude extracts were centrifuged at 13,000 rpm at 4 °C for 10 min to pellet cell debris. Supernatants were collected, and total protein concentrations were quantified by the Bradford protein assay (Bradford, 1976) using bovine serum albumin (BSA; Sigma Aldrich) as a standard.

As the normalized amount of fresh and lyophilized leaves and chloroplasts was used for protein extraction, we loaded gels on the basis of an equal amount of protein extract for Western blotting. About 50 µg of total soluble protein from each sample was boiled in sample buffer and separated by a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in reducing conditions. Separated proteins were electro–transferred onto a nitrocellulose membrane (Bio-Rad) using Mini TransBlot (Bio-Rad), following the manufacturer’s instructions. Chloroplast-made DARPin G3 was detected using rabbit anti-His-tag antibody as a primary antibody and goat anti-rabbit conjugated with horseradish peroxidase (HRP) antibody as a secondary antibody. The bands were detected with the addition of a DAB-peroxidase substrate solution.

ELISA quantification of chloroplast-made DARPin G3 proteins

An enzyme-linked immunosorbent assay (ELISA) was used to measure the amounts of chloroplast-made DARPin G3 expression in fresh and lyophilized leaves and fresh and lyophilized chloroplasts of transplastomic and wild-type tobacco plants. About 50 µg of total soluble proteins in extraction buffer (PBS 1X, pH 7.4, 150 mM NaCl, and 1X EDTA-free protease inhibitor) from fresh and lyophilized materials were bound to a 96-well polyvinyl chloride microtiter plate overnight at 4 °C. Background was blocked for two hours at 37 °C with 300 µl of blocking buffer (1% BSA (w/v) in 1X PBS buffer containing 0.1% Tween 20 (w/v)). Wells were incubated for 2 h at 37 °C with rabbit anti-His-tag antibody diluted to 1:1000 with blocking buffer as the primary antibody, and then three times washed with PBS-T (PBS buffer containing 0.1% Tween 20 (w/v)). The blots were then incubated with a goat anti-rabbit antibody conjugated with HRP in blocking buffer at a dilution of 1:10000 as a secondary antibody for 2 h at 37 °C. The plate was washed again as stated above and developed using the 3, 3, 5, 5-tetramethylbenzidine (TMB) peroxidase substrate solution (200 mM citrate buffer, pH 3.95, 1% TMB, 0.01% H2O2). The reaction was terminated with 100 µl of 2 M H2SO4, and the optical density of each well was measured using an ELISA reader at 450 nm. For a standard curve, purified 15 kDa His-tagged standard protein was added in a serial dilution ranging from 3 to 25 ng/ml to the microplate and processed as described above. The standard curve was used to quantify the amount of DARPin G3 protein in the total soluble proteins that remained in freeze-dried materials compared to fresh tissue. The yield of DARPin G3 was calculated and expressed as a percentage of total soluble protein (TSP) and as the amount of DARPin G3 protein (mg/g).

Affinity purification of chloroplast-made DARPin G3

The chloroplast-made DARPin G3 was purified directly from total soluble protein extracted from fresh and lyophilized leaves and chloroplasts of transplastomic tobacco plants using the QIAexpress Ni-NTA Protein Purification System (QIAGEN). Total soluble proteins were mixed with 4x binding buffer (2 M NaCl and 2X PBS, no imidazole) at a 4:1 ratio in batch mode and incubated for 2 h at 4 °C. For the washing and elution steps, the protein–resin complex was packed into a column. The column was then washed with the wash buffer (0.5 M NaCl and 0.5X PBS, no imidazole) and eluted in 0.5 mL fractions for three times with the elution buffer (0.5 M NaCl and 0.5X PBS containing 200 mM imidazole). The eluted fractions were dialyzed against PBS three times, aliquoted, and stored at − 20 °C. SDS-PAGE and Coomassie blue staining were performed to detect chloroplast-made DARPin G3.

HER2 receptor binding assay

A binding test was carried out according to our previously described method [19] to measure the affinity of chloroplast-made DARPin G3 from lyophilized materials of transplastomic tobacco plants for the extracellular domain (ECD) of HER2 in 96-well pre-coated plates with the HER2-ECD. 100 µL of 1 g/mL HER2-ECD (Sino Biological, 10,004-HCCH) were used for the coating, which was then kept at 4 °C overnight. After being washed twice with phosphate-buffered saline solution containing 0.1% tween-20 (PBS-T), the plate was blocked of non-specific binding sites on a shaker for 1 h at room temperature with PBS-T containing 1% BSA. The ELISA procedure used a serial dilution ranging from 10 to 100 nM of purified chloroplast-made DARPin G3 in PBS-T/BSA, which was applied to triplicate wells in 100 µL volumes and incubated at room temperature for 1 h with shaking. After that, each well was washed three times with 200 µL of PBS-T. A rabbit anti-His-tag antibody (1:1000 in PBS-T/BSA), which recognizes the N-terminal histidine-glutamate (HE)3-tag of the chloroplast-made DARPin G3, was used to probe the binding and was incubated for an hour at room temperature on a shaker. After three washes with PBS-T, the goat anti-rabbit antibody conjugated with HRP was incubated for one hour at room temperature on a shaker in a final volume of 100 µL of PBS-T/BSA at a dilution of 1:10000. Following this, each well was washed with 200 µL of PBS-T for triplets. 100 µL of TMB solution was used to create the ELISA, which was then incubated at room temperature until a satisfactory colorimetric shift was seen. The reaction was stopped by adding 100 µL of 2 N H2SO4, and absorbance readings were measured at 450 nm using an ELISA plate reader. A total soluble protein of wild-type plants and bovine serum albumin (BSA) served as negative controls.

Cell culture conditions

We examined the HER2 specificity of chloroplast-made DARPin G3 from fresh and lyophilized leaves and chloroplasts using two distinct breast cancer cell lines that express HER2 to varying extents. SKBR-3 cells, a highly expressed HER2 breast cancer cell line, and MDA-MB-231 cells, which hardly express HER2, were provided by the Immunology Research Center (Tabriz, Iran). SKBR-3 and MDA-MB-231 cells were cultured in cell culture flasks containing RPMI 1640 medium supplemented with 1% penicillin (10,000 units/ml), 1% streptomycin (10 mg/ml), and 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator with 5% CO2.

Flow cytometry

The binding specificity of the chloroplast-made DARPin G3 from fresh and lyophilized plant materials to HER2 on breast carcinoma cell lines was examined via flow cytometry by following the procedures as described by us [19]. In brief, two distinct cancer cell lines were individually prepared. After removing the medium, the cells were treated for 10 min with 5 ml of 0.2% EDTA. The cells were then transferred to tubes and centrifuged for 5 min at 4 °C at 1000 rpm. After removing the EDTA-containing supernatant, 5 ml of fresh medium was added. 1 ml of cells counted and diluted to 106 cells per ml were used for each test condition. After washing the cells with cold PBS containing 1% BSA, 100 nM chloroplast-derived DARPin G3 was added to the cells and incubated at 4 °C for 1 h. Following a cold PBS/BSA wash, cells were incubated for 1 h at 4 °C with 200 µl of rabbit anti-His-tag antibody diluted in PBS/BSA (1:1000). The cells were then washed with cold PBS/BSA, followed by incubation for 30 min at 4 °C in the presence of 200 µl of fluorescein isothiocyanate-coupled donkey (FITC) anti-rabbit antibody at a dilution of 1:1000. After washing with cold PBS, the cells were resuspended in 500 µl of cold PBS and analyzed on a MACSQuant 10 Flow Cytometer (Miltenyi Biotec, Germany) at a flow rate of 500 s−1. Fluorescence was detected at 525 nm after being excited with an argon laser at 488 nm. Cells were then gated according to size scatter, forward scatter, and pulse width, so only single cells were analyzed. Negative groups included cells that had not been treated or that had been treated with anti-His tag antibody followed by FITC donkey anti-rabbit IgG. A total of 10,000 cell events were recorded per sample. Fluorescence data was analyzed using the software FlowJo (Tree Star, Ashland, OR). Cells treated with PBS or antibodies in the absence of chloroplast-made DARPin G3 were used as controls.

Immunofluorescent microscopy

To begin, cells were seeded into sterile 96-well culture plates at a density of 1.0 × 104 in RPMI 1640 growth media and incubated at 37 °C and 5% CO2 for the duration of the night. After removing the medium, the cells were washed three times with ice-cold PBS containing 1% BSA. Cells were treated for 1 h at 4 °C with 200 nM chloroplast-made DARPin G3 in PBS/BSA. Following three washes with cold PBS/BSA, the cells were incubated for 1 h at 4 °C with rabbit anti-His-tag antibody (1:1000 diluted in PBS/BSA) as the primary antibody. Cells were washed and incubated for 30 min at 4 °C in the dark with FITC donkey anti-rabbit IgG (1:1000 diluted in PBS/BSA) as a secondary antibody. After three washes with cold PBS, cells were analyzed using the Citation 5 Cell Imaging Multimode Reader (BioTek, Winooski, VT). The laser at 469 nm excited the DARPin G3-FITC, and the fluorescence of FITC was registered at 525 nm.