The lyophilized chloroplasts store synthetic DARPin G3 as bioactive encapsulated organelles.

BACKGROUND: The high cost of fermentation, purification, cold storage and transportation, short shelf life, and sterile delivery methods of biopharmaceuticals, is a matter for producers and consumers as well. Since the FDA has now approved plant cells for large-scale, cost-effective biopharmaceutical production, the isolation and lyophilization of transplastomic chloroplasts can cover concerns about limitations. DARPins are engineered small single-domain proteins that have been selected to bind to HER2 with high affinity and specificity. HER2 is an oncogene involved in abnormal cell growth in some cancers and the target molecule for cancer immunotherapy. RESULTS: In this study, we reported the prolonged stability and functionality of DARPin G3 in lyophilized transplastomic tobacco leaves and chloroplasts. Western blot analysis of lyophilized leaves and chloroplasts stored at room temperature for up to nine months showed that the DARPin G3 protein was stable and preserved proper folding. Lyophilization of leaves and isolated chloroplasts increased DARPin G3 protein concentrations by 16 and 32-fold, respectively. The HER2-binding assay demonstrated that the chloroplast-made DARPin G3 can maintain its stability and binding activity without any affinity drop in lyophilized leaf materials throughout this study for more than nine months at room temperature. CONCLUSION: Lyophilization of chloroplasts expressing DARPin G3 would further reduce costs and simplify downstream processing, purification, and storage. Compressed packages of lyophilized chloroplasts were much more effective than lyophilized transplastomic leaves considering occupied space and downstream extraction and purification of DARPin G3 after nine months. These methods facilitate any relevant formulation practices for these compounds to meet any demand-oriented needs.

The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology.

BACKGROUND: Designed mimetic molecules are attractive tools in biopharmaceuticals and synthetic biology. They require mass and functional production for the assessment of upcoming challenges in the near future. The DARPin family is considered a mimetic pharmaceutical peptide group with high affinity binding to specific targets. DARPin G3 is designed to bind to the HER2 (human epidermal growth factor receptor 2) tyrosine kinase receptor. Overexpression of HER2 is common in some cancers, including breast cancer, and can be used as a prognostic and predictive tool for cancer. The chloroplasts are cost-effective alternatives, equal to, and sometimes better than, bacterial, yeast, or mammalian expression systems. This research examined the possibility of the production of the first antibody mimetic, DARPin G3, in tobacco chloroplasts for HER2 imaging in oncology. RESULTS: The chloroplast specific DARPin G3 expression cassette was constructed and transformed into N. tabacum chloroplasts. PCR and Southern blot analysis confirmed integration of transgenes as well as chloroplastic and cellular homoplasmy. The Western blot analysis and ELISA confirmed the production of DARPin G3 at the commercial scale and high dose with the rate of 20.2% in leaf TSP and 33.7% in chloroplast TSP. The functional analysis by ELISA confirmed the binding of IMAC purified chloroplast-made DARPin G3 to the extracellular domain of the HER2 receptor with highly effective picomolar affinities. The carcinoma cellular studies by flow cytometry and immunofluorescence microscopy confirmed the correct functioning by the specific binding of the chloroplast-made DARPin G3 to the HER2 receptor on the surface of HER2-positive cancer cell lines. CONCLUSION: The efficient functional bioactive production of DARPin G3 in chloroplasts led us to introduce plant chloroplasts as the site of efficient production of the first antibody mimetic molecules. This report, as the first case of the cost-effective production of mimetic molecules, enables researchers in pharmaceuticals, synthetic biology, and bio-molecular engineering to develop tool boxes by producing new molecular substitutes for diverse purposes.

The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology

BACKGROUND: Designed mimetic molecules are attractive tools in biopharmaceuticals and synthetic biology. They require mass and functional production for the assessment of upcoming challenges in the near future. The DARPin family is considered a mimetic pharmaceutical peptide group with high affinity binding to specific targets. DARPin G3 is designed to bind to the HER2 (human epidermal growth factor receptor 2) tyrosine kinase receptor. Overexpression of HER2 is common in some cancers, including breast cancer, and can be used as a prognostic and predictive tool for cancer. The chloroplasts are cost-effective alternatives, equal to, and sometimes better than, bacterial, yeast, or mammalian expression systems. This research examined the possibility of the production of the first antibody mimetic, DARPin G3, in tobacco chloroplasts for HER2 imaging in oncology. RESULTS: The chloroplast specific DARPin G3 expression cassette was constructed and transformed into N. tabacum chloroplasts. PCR and Southern blot analysis confirmed integration of transgenes as well as chloroplastic and cellular homoplasmy. The Western blot analysis and ELISA confirmed the production of DARPin G3 at the commercial scale and high dose with the rate of 20.2% in leaf TSP and 33.7% in chloroplast TSP. The functional analysis by ELISA confirmed the binding of IMAC purified chloroplast-made DARPin G3 to the extracellular domain of the HER2 receptor with highly effective picomolar affinities. The carcinoma cellular studies by flow cytometry and immunofluorescence microscopy confirmed the correct functioning by the specific binding of the chloroplast-made DARPin G3 to the HER2 receptor on the surface of HER2-positive cancer cell lines. CONCLUSION: The efficient functional bioactive production of DARPin G3 in chloroplasts led us to introduce plant chloroplasts as the site of efficient production of the first antibody mimetic molecules. This report, as the first case of the cost-effective production of mimetic molecules, enables researchers in pharmaceuticals, synthetic biology, and bio-molecular engineering to develop tool boxes by producing new molecular substitutes for diverse purposes.

Computer-Based Tools Unmask Critical Mineral Nutrient Interactions in Hoagland Solution for Healthy Kiwiberry Plant Acclimatization.

The aim of this study was to better understand the response of ex vitro acclimatized plants grown to a set of mineral nutrient combinations based on Hoagland solution. To reach that, two computer-based tools were used: the design of experiments (DOE) and a hybrid artificial intelligence technology that combines artificial neural networks with fuzzy logic. DOE was employed to create a five-dimensional IV-design space by categorizing all macroelements and one microelement (copper) of Hoagland mineral solution, reducing the experimental design space from 243 (35) to 19 treatments. Typical growth parameters included hardening efficiency (Hard), newly formed shoot length (SL), total leaf number (TLN), leaf chlorophyll content (LCC), and leaf area (LA). Moreover, three physiological disorders, namely, leaf necrosis (LN), leaf spot (LS), and curled leaf (CL), were evaluated for each treatment (mineral formulation). All the growth parameters plus LN were successfully modeled using neuro-fuzzy logic with a high train set R 2 between experimental and predicted values (72.67 < R 2 < 98.79). The model deciphered new insights using different sets of "IF-THEN" rules, pinpointing the positive role of Mg2+ and Ca2+ to improve Hard, SL, TLN, and LA and alleviate LN but with opposite influences on LCC. On the contrary, TLN and LCC were negatively affected by the addition of NO3 - into the media, while NH4 + in complex interaction with Cu2+ or Mg2+ positively enhanced SL, TLN, LCC, and LA. In our opinion, the approach and results achieved in this work are extremely fruitful to understand the effect of Hoagland mineral nutrients on the healthy growth of ex vitro acclimatized plants, through identifying key factors, which favor growth and limit physiological abnormalities.

Cystatins may confer viral resistance in plants by inhibition of a virus-induced cell death phenomenon in which cysteine proteinases are active: cloning and molecular characterization of a cDNA encoding cysteine-proteinase inhibitor (celostatin) from Celosia cristata (crested cock's comb).

Cystatins (cysteine proteinase inhibitors) have been recently used in plants as antiviral strategy against those viruses whose replication involves cysteine proteinase activity. We proposed an idea that cystatins may confer resistance by inhibition of a virus-induced cell-death phenomenon in which cysteine proteinases are active. To test this idea, a full-length cDNA library was constructed from the preflowering stage of Celosia cristata (crested cock's comb) leaves, and a cDNA clone with cystatin domain was isolated using an oligonucleotide probe designed on the basis of the conserved peptide of plant cystatins. It was expressed in an Escherichia coli expression system as a fusion protein. The purified recombinant product, termed 'celostatin' (Celosia cystatin), inhibited the enzymatic activity of papain indicating its cystatin activity and prevented TMV (tobacco mosaic virus)-induced hypersensitive-response cell death in Nicotiana glutinosa (a wild species of tobacco) leaves by 65-70% at the concentration of approx. 50 ng/ml. It also offered resistance against TMV and caused normal growth of the test plant. Since the activity of cysteine proteinases is not involved in the TMV replication process, we speculated that inhibition of the hypersensitive response by celostatin may be due to the inactivation of proteolysis involved in the plant cell death programme, a phenomenon that has already been reported in animal systems.