Recombinant production and characterization of the integrin-binding LERGDT peptide

Abstract

Chronic kidney disease (CKD) remains a global health burden, necessitating the development of scalable therapeutic interventions. While the LERGDT peptide has previously demonstrated integrin-binding and anti-fibrotic activity, its transition toward clinical applications is hindered by the high cost of large-scale chemical synthesis. This study presents a robust bioprocess for the recombinant production of a 20-repeat tandem LERGDT construct (PEP-1) using an Escherichia coli expression system. To ensure structural stability and protection against proteolytic degradation, PEP-1 was engineered as a fusion protein with N-terminal ketosteroid isomerase (KSI) and a C-terminal six-histidine tag. In silico analysis using AlphaFold and HADDOCK predicted high structural stability and specific binding affinity to integrins. The bioprocess system achieved a significant total protein yield of 52.94 mg.L^-1 , with the fused protein purified via nickel-affinity chromatography and successfully cleaved into functional fragments (hPEP-1) using hydroxylamine hydrolysis. Product identity was confirmed by means of SDS-PAGE at ~35 kDa and validated by using tandem LC-MS. ELISA-based assays confirmed that both the fusion protein and released fragments maintained specific integrin-binding functionality. By establishing a high-yield, reproducible production protocol, this work provides a cost-effective alternative to synthetic methods, facilitating large-scale testing required for peptide-based CKD therapeutics. This scalable bioprocess accelerates the translation of LERGDT-based interventions from bench to bedside, enhancing the accessibility of affordable long-term treatments for CKD patients.