جستجوی مقالات مرتبط با کلیدواژه « chloroplast transformation » در نشریات گروه « بیوتکنولوژی و ژنتیک گیاهی »

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.

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.

Plants are suitable replacements for current biodrugs and recombinant protein expression systems such as transgenic animals or microbial systems. As the plant plastid genome is highly polyploid, the transformation of chloroplast permits the introduction of thousands of copies of foreign gene per plant cell and generates extraordinarily high levels of recombinant protein. Cardiovascular diseases are the second one leading cause of human mortality after cancer. Human tissue-type plasminogen activator (tPA) is one of the most important pharmaceutical recombinant proteins involved in the breakdown of blood clots in different parts of body such as brain and heart blood vessels. The truncated form of tissue plasminogen activator (K2S) has longer plasma half life, better diffusion into the clot and higher fibrinolytic activity. In this study, in order to introduction of K2S gene in tobacco chloroplast, after construct design, the interest gene was ligated to pKCZ vector and cloned in E. coli. Following the successfully shooting of the K2S-containing vector (pKCZK2S) to leaf explants using the biolistic delivery procedure, the explants were selected on selection medium containing 500 mgL-1 spectinomycine antibiotic. After regeneration of grown shoots on selective medium, in order to achieve homoplasmy, fourth rounds of selection and regeneration in presence of spectinomycine antibiotic were performed. The presence, site-specific integration, expression of the K2S gene and homoplasmy in transplastomic plants were confirmed by PCR, RT-PCR and Southern blotting methods.