DESIGN AND IN VITRO FUNCTIONAL EVALUATION OF M-6HIS-GLP-1-6HIS, A DUAL-HEXAHISTIDINE-TAGGED GLP-1 VARIANT

Introduction: Native GLP-1 has a short half-life due to rapid enzymatic degradation, limiting therapeutic application. Sequence engineering may facilitate peptide production and purification but requires functional validation. We designed M-6HIS-GLP-1-6HIS, a recombinant GLP-1(7–37) variant carrying N- and C-terminal hexahistidine tags for affinity purification, and evaluated it in pancreatic β-cells.

Methods: M-6HIS-GLP- 1-6HIS was designed using sequence analysis. Structural modeling and analysis were performed with AlphaFold 3 and ChimeraX. A codon-optimized sequence was cloned into pET32(a), expressed in E. coli BL21(DE3) after 200 µM IPTG induction, and purified by Ni²⁺-affinity chromatography. MIN6 cells served as a β-cell model. Glp1r expression was confirmed by qPCR. Cells were exposed to peptide (100 nM) for 24 h in glucose-free medium or medium containing 25 mM glucose. Metabolic activity, insulin secretion, and mitochondrial activity were assessed by MTT assay, ELISA, and fluorescence microscopy, respectively.

Results: Structural modeling predicted preservation of key peptide–GLP-1R contacts and an increased peptide–receptor interaction for M-6HIS-GLP-1-6HIS. In the MTT assay, cellular metabolic activity in peptide-treated MIN6 cells was 5.6% higher in the presence of 25 mM glucose than under glucose-free conditions. Insulin secretion increased in the presence of 25 mM glucose compared with glucose-free conditions (p<0.01) and with the untreated control (p<0.05). Mitochondrial fluorescence showed a distinct condition-dependent pattern. Under glucose-free conditions, M-6HIS-GLP-1-6HIS reduced relative MitoTracker fluorescence per cell compared with the untreated control (p=0.0044). In peptide-treated cells, mitochondrial fluorescence increased in 25 mM glucose and was comparable to the untreated high-glucose control (p=0.79).

Conclusions: M-6HIS-GLP-1-6HIS demonstrated a glucose-dependent insulin secretory response and glucose-associated changes in cellular metabolic and mitochondrial readouts in MIN6 cells. These findings support further functional investigation but do not establish GLP-1R-mediated agonism or DPP-4 resistance.