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.

Chloroplast Transformation in Arabidopsis.

Plastids (chloroplasts) are the defining organelles of plants and eukaryotic algae. In addition to performing photosynthesis, plastids harbor numerous other metabolic pathways and therefore are often referred to as the biosynthetic center of the plant cell. The chloroplasts of seed plants possess dozens of copies of a circular genome of ∼150 kb that contains a conserved set of 120 to 130 genes. The engineering of this genome by genetic transformation is technically challenging and currently only possible in a small number of species. In this article, we describe the methods involved in generating stable chloroplast-transformed (transplastomic) plants in the model species Arabidopsis (Arabidopsis thaliana). The protocols presented here can be applied to (1) target genes in the Arabidopsis chloroplast genome by reverse genetics and (2) express reporter genes or other foreign genes of interest in plastids of Arabidopsis plants. © 2021 The Authors. Basic Protocol 1: Generation of root-derived microcallus material for biolistic transformation Basic Protocol 2: Chloroplast transformation by biolistic bombardment of root-derived microcalli Basic Protocol 3: Regeneration of transplastomic lines and seed production.

Evaluation of lettuce chloroplast and soybean cotyledon as platforms for production of functional bone morphogenetic protein 2.

The bone morphogenetic protein BMP2 plays a crucial role in the formation and regeneration of bone and cartilage, which is critical for maintaining skeletal integrity and bone fracture repair. Because of its important role in osteogenic properties it has been commercially produced for clinical use. Here we report attempts to express human BMP2 using two plant systems (lettuce chloroplast and soybean seeds). The rhBMP2 gene (coding for the 13 kDa active polypeptide) was introduced in two regions of the lettuce chloroplast genome. Two homoplasmic events were achieved and RT-PCR demonstrated that the BMP2 gene was transcribed. However, it was not possible to detect accumulation of rhBMP2 in leaves. Two soybean events were achieved to express a full-length hBMP2 gene (coding for the 45 kDa pro-BMP2) fused with the α-coixin signal peptide, under control of the ß-conglycinin promoter. Pro-BMP2 was expressed in the transgenic seeds at levels of up to 9.28% of the total soluble seed protein as determined by ELISA. It was demonstrated that this recombinant form was biologically active upon administration to C2C12 cell cultures, because it was able to induce an osteogenic cascade, as observed by the enhanced expression of SP7 (osterix) and ALPI (alkaline phosphatase) genes. Collectively, these results corroborated our previous observation that soybean seeds provide an effective strategy for achieving stable accumulation of functional therapeutic proteins, such as BMP2.

Chikungunya virus vaccines: Current strategies and prospects for developing plant-made vaccines.

Chikungunya virus is an emerging pathogen initially found in East Africa and currently spread into the Indian Ocean Islands, many regions of South East Asia, and in the Americas. No licensed vaccines against this eminent pathogen are available and thus intensive research in this field is a priority. This review presents the current scenario on the developments of Chikungunya virus vaccines and identifies the use of genetic engineered plants to develop attractive vaccines. The possible avenues to develop plant-made vaccines with distinct antigenic designs and expression modalities are identified and discussed considering current trends in the field.