Design and Expression of mGLP1-DARPin-Pen Trivalent Protein with Oral Potential for Type 2 Treatment in Tobacco Chloroplasts

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.