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

Shammi Akter, Shajia Afrin, Jaeyoon Kim, Joohyun Kang, Md Abdur Razzak, Per-Olof Berggren, Inhwan Hwang

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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.

Original languageEnglish
Article number1062658
JournalFrontiers in plant science
Volume13
DOIs
Publication statusPublished (in print/issue) - 22 Dec 2022

Bibliographical note

Funding Information:
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No.2022R1A2C1091844). JK was supported by a National Research Foundation of Korea (NRF) grant (2021R1I1A1A01051391).

Publisher Copyright:
Copyright © 2022 Akter, Afrin, Kim, Kang, Razzak, Berggren and Hwang.

Keywords

  • Exendin-4
  • type-2 diabetics
  • enterokinase
  • insulin
  • recombinant protein production

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