Production of active Exendin-4 in Nicotiana benthamiana and its application in treatment of type-2 diabetics

Abstract

GLP-1 (Glucagon-like peptide-1) is a peptide that stimulates insulin secretion from the β-cell for glycemic control of the plasma blood glucose level. Its mimetic exenatide (synthetic Exendin-4) with a longer half-life of approximately 3.3–4 h is widely used in clinical application to treat diabetes. Currently, exenatide is chemically synthesized. In this study, we report that the GLP-1 analogue recombinant Exendin-4 (Exdn-4) can be produced at a high level in Nicotiana benthamiana, with an estimated yield of 50.0 µg/g fresh biomass. For high-level expression, we generated a recombinant gene, B:GB1:ddCBD1m:8xHis : Exendin-4 (BGC : Exdn-4), for the production of Exendin-4 using various domains such as the BiP signal peptide, the GB1 domain (B1 domain of streptococcal G protein), a double cellulose binding domain 1 (CBD1), and 8 His residues (8xHis) to the N-terminus of Exendin-4. GB1 was used to increase the expression, whereas double CBD1 and 8xHis were included as affinity tags for easy purification using MCC beads and Ni2+-NTA resin, respectively. BGC : Exdn-4 was purified by single-step purification to near homogeneity using both Ni2+-NTA resin and microcrystalline cellulose (MCC) beads. Moreover, Exdn-4 without any extra residues was produced from BGC : Exdn-4 bound onto MCC beads by treating with enterokinase. Plant-produced Exdn-4 (Exendin-4) was as effective as chemically synthesized Exendin-4 in glucose-induced insulin secretion (GIIS) from mouse MIN6m9 cells a pancreatic beta cell line.

Production of Mature Recombinant Human Activin A in Transgenic Rice Cell Suspension Culture

“…Previous studies have demonstrated the production of human TGFβ1 in Nicotiana benthamiana, the mature form with biological activity obtained after enterokinase treatment [38]. Based on the specific cleavage property of enterokinase, the fusion protein is released after treatment with this enzyme [39,40]. In this study, to produce bioactive activin A, a cleavage site of enterokinase (D4K) was introduced between the pro-region and mature region of rhAA.…”

Peptides Are Cardioprotective Drugs of the Future: The Receptor and Signaling Mechanisms of the Cardioprotective Effect of Glucagon-like Peptide-1 Receptor Agonists

“…These findings demonstrate that enzyme-resistant GLP-1 analogs could cross the BBB. The half-life of exendin-4 is 2.4-4 h, allowing it to penetrate into the brain after intravenous administration [66,67]. It was found that exendin-4 is soluble in water but exhibits moderate lipophilicity, allowing it to quickly penetrate into the brain after intravenous injection regardless of circumventricular organs which are devoid of the BBB [68].…”