Ultra-compact amyloidogenic fusion tags mediated efficient production of liraglutide precursors and molecular insights

Abstract The recombinant production of therapeutic peptides such as the glucagon-like peptide-1 (GLP-1) analogue liraglutide precursor in E. coli is often constrained by peptide instability, susceptibility to intracellular proteolysis, and the use of large fusion partners that reduce the target peptide mass fraction and impose a metabolic burden on the host. Here, we developed a minimalist fusion strategy employing ultra-compact short amyloidogenic peptides (SAPs) as multifunctional fusion tags that consolidate aggregation-inducing, affinity-capture, and protease-cleavage functionalities within a footprint of < 3 kDa. Twelve codon-optimized constructs encoding the liraglutide precursor [Arg³⁴-GLP-1(7–37)] were engineered using N-terminal SAP motifs IVFK, KLVFF, VQIVYK, or VQTIVFQ in one, two, or three GPG-linked tandem repeats, each followed by a His₆ tag and TEV protease cleavage site. Eight constructs produced detectable fusion proteins in E. coli BL21(DE3), with expression strongly dependent on SAP sequence and repeat number. VQTIVFQ variants achieved the highest overall expression, with VQTIVFQ-3× reaching 115 ± 23 mg·L⁻¹. The highest mean calculated liraglutide precursor yields were obtained with IVFK-1 × (53 ± 3 mg·L⁻¹) and VQTIVFQ-1 × (52 ± 9 mg·L⁻¹). Statistical analysis confirmed construct-dependent differences in fusion protein yield, calculated precursor yield, and insoluble fraction. All expressing constructs localized the fusion protein predominantly to inclusion bodies, with insoluble fraction values ranging from 65 ± 3% to 100 ± 0%. Purification by immobilized metal affinity chromatography, followed by TEV protease cleavage, released the target peptide, whose molecular mass and amino acid sequence were confirmed by LC-MS analysis. Scanning electron microscopy and Thioflavin-T fluorescence assays further revealed β-sheet-rich fibrillar assemblies characteristic of amyloid-like aggregates, consistent with the SAP-mediated inclusion body formation observed in E. coli . Computational prediction using PASTA 2.0 supported repeat-dependent increase in β-sheet-forming propensity across SAP fusion designs. Collectively, these results establish that ultra-compact SAP-His₆-TEVcs fusion tags enable inclusion body-based expression, affinity purification, and precise proteolytic release of the liraglutide precursor in E. coli , providing a basis for further development of compact amyloidogenic tags for recombinant production of small therapeutic peptides.