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Showing 2 results for Transplastomic
Maryam Ehsasatvatan, Dr Bahram Baghban Kohnehrouz,
Volume 9, Issue 2 (3-2023)
Abstract
Plastid engineering gives numerous benefits for the next generation of transgenic technology, consisting of the convenient use of transgene stacking and the production of high expression levels of recombinant proteins. Designed ankyrin repeat proteins (DARPin) are relatively small non-immunoglobulin scaffold proteins that bind to their specific target with high affinity. The G3 is a type of DARPin designed to bind to the HER2 tyrosine kinase receptor (human epidermal growth factor receptor 2). We previously developed a bioprocess for the production of DARPin G3 in tobacco chloroplasts as an imaging agent in HER2 over-expressed cancers. In this study, we analyzed the expression and homoplasmic stability of DARPin G3 gene in vegetative and generative T1 generation of transplastomic tobacco plants. The presence of DARPin G3 gene in the next generation of transplastomic plants was confirmed with specific primers by PCR analysis. Southern blot analysis confirmed the homoplasmic status of transplastomic plants. The western blot analysis confirmed the accumulation of the DARPin G3 in the chloroplasts of next generation of transplastomic plants. The DARPin G3 protein content was estimated around 33% by ELISA in chloroplast total soluble protein (TSP) of the transplastomic plants. Results confirmed that the DARPin G3 gene in vegetative and generative T1 generation of transplastomic tobacco plants was stably and highly expressed.
Maryam Ehsasatvatan, Bahram Baghban Kohnehrouz,
Volume 10, Issue 2 (2-2024)
Abstract
The global prevalence of type 2 diabetes mellitus is continuously increasing, and there is currently no definitive cure for type 2 diabetes. The potent glucagon-like peptide 1 (GLP-1), a natural small incretin hormone, enhances insulin secretion in a glucose-dependent manner. However, the exceedingly short half-life of GLP-1 limits its therapeutic applications. Albumin-binding DARPin can be used to increase the serum half-lives of therapeutic proteins, peptides, and small compounds. In this study, a long-acting GLP-1 agonist with oral delivery potential containing a protease-resistant GLP-1, an albumin-binding DARPin, and Penetratin as a fusion protein was expressed in a bioencapsulated form within tobacco chloroplasts to confer digestive system protection in plant cells. The successful transformation of tobacco chloroplasts with trivalent fusion protein-coding genes was conducted using a pPRV111A chloroplastic expression vector and a gene gun. Homoplasmic transplastomic plants were obtained after three rounds of selection in selection medium containing 500 mg/L spectinomycin and streptomycin. Transgene integration and homoplasmic status in the transplastomic plants were confirmed by PCR and Southern blot analyses. Western blot analysis confirmed the accumulation of the mGLP1-DARPin-Pen fusion protein in the chloroplasts of the transplastomic plants. The fusion protein content estimated by ELISA was 21.8% of the total soluble protein content in the transplastomic plants. The successful expression of the designed fusion protein indicated that the production of functional GLP-1 in plants may facilitate the development of a low-cost, orally deliverable form of this protein for the treatment of type 2 diabetes.
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