A new trivalent recombinant protein for type 2 diabetes mellitus with oral delivery potential: design, expression, and experimental validation

Abstract

Glucagon-like peptide-1 (GLP-1) receptor agonists are increasingly used in clinical practice for the management of type 2 diabetes mellitus. However, the extremely short half-life of GLP-1 and the need for subcutaneous administration limit its clinical application. Thus, half-life extension and alternative delivery methods are highly desired. DARPin domains with high affinity for human serum albumin (HSA) have been selected for the half-life extension of therapeutic peptides and proteins. In the present study, novel trivalent fusion proteins as long-acting GLP-1 receptor agonists with potential for oral delivery were computationally engineered by incorporating a protease-resistant modified GLP-1, an anti-human serum albumin DARPin, and an approved cell-penetrating peptide (Penetratin, Tat, and Polyarginine) linked either by rigid or flexible linkers. Theoretical studies and molecular dynamics simulation results suggested that mGLP1-DARPin-Pen has acceptable quality and stability. Moreover, the potential affinity of the selected fusion proteins for GLP-1 receptor and human serum albumin was explored by molecular docking. The recombinant construct was cloned into the pET28a vector and expressed in Escherichia coli. SDS-PAGE analysis of the purified fusion protein matched its molecular size and was confirmed by western blot analysis. The results demonstrated that the engineered fusion protein could bind HSA with high affinity. Importantly, insulin secretion assays using a mouse pancreatic β-cell line (β-TC6) revealed that the engineered trivalent fusion protein retained the ability to stimulate cellular insulin secretion. Immunofluorescence microscopy analysis indicated the CPP-dependent cellular uptake of mGLP1-DARPin-Pen. These findings demonstrated that mGLP1-DARPin-Pen is a highly potent oral drug candidate that could be particularly useful in the treatment of type 2 diabetes mellitus.

Communicated by Ramaswamy H. Sarma

Keywords:

• Cell-penetrating peptide
• DARPin
• fusion protein
• glucagon-like peptide 1
• human serum albumin
• molecular dynamic simulation
• type 2 diabetes mellitus

Data availability statement

The dataset supporting the conclusions of this article is included within the article and its additional file. Further inquiries cannot be shared temporally, as the data also forms part of an ongoing study.

Authors’ contributions

M. E., designed the study and experiments, performed the experiments, analyzed the data, and wrote the manuscript. B. B. K., conceived, designed, organized, and supervised the study, provided specialized scientific and technical support, and revised the manuscript. All authors read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This research is supported by the Research Grant of the University of Tabriz (number S/4829-14020128).