Protein and peptide letters · 2025
Shepherin II Gene Synthesis and Peptide Characterization: E. coli Expression, Purification, and Antiviral Activity.
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Abd Elfattah Azza, Samir Safia, Okasha Hend, Atef Azza Ahmed, Taha Alshaimaa
Аннотация
INTRODUCTION: The shepherin II peptide is characterized by a histidine/glycine-rich sequence. This study aimed to design, express recombinantly, and evaluate the antiviral activity of shepherin II against hepatitis A virus (HAV).
METHODS: The shepherin II gene was reverse-translated, cloned into the pET-3a vector, and expressed in E. coli BL21 (DE3) pLysS cells induced with 2 mM IPTG. Purification was achieved via cation exchange chromatography, and intact mass analysis using mass spectrometry was carried out. Cytotoxicity on normal Vero cells and antiviral activity on HAV were evaluated.
RESULTS: The mass spectrometry confirmed a primary peptide fragment with a molecular weight of 3,421.30 Da (100% relative abundance). SDS-PAGE verified peptide expression. Cytotoxicity tests on Vero cells showed a CC50 of 219.26 ± 7.91 μg/ml. Antiviral assay revealed an EC50 of 113.92 ± 4.58 μg/ml against HAV, resulting in a selectivity index (SI) of 1.92. This SI indicates limited selectivity compared to the reference drug amantadine, which exhibited an EC50 of 5.67 ± 0.71 μg/ml and an SI of 53.41.
DISCUSSION: The recombinant expression of shepherin II was successfully achieved and confirmed by mass spectrometry and SDS-PAGE. The peptide showed measurable antiviral activity against HAV.
CONCLUSION: This study demonstrated the feasibility of recombinant shepherin II production and assessed its antiviral activity. However, the limited selectivity index of shepherin II remains a challenge that needs to be addressed through molecular modification or alternative delivery strategies to improve its clinical potential.
METHODS: The shepherin II gene was reverse-translated, cloned into the pET-3a vector, and expressed in E. coli BL21 (DE3) pLysS cells induced with 2 mM IPTG. Purification was achieved via cation exchange chromatography, and intact mass analysis using mass spectrometry was carried out. Cytotoxicity on normal Vero cells and antiviral activity on HAV were evaluated.
RESULTS: The mass spectrometry confirmed a primary peptide fragment with a molecular weight of 3,421.30 Da (100% relative abundance). SDS-PAGE verified peptide expression. Cytotoxicity tests on Vero cells showed a CC50 of 219.26 ± 7.91 μg/ml. Antiviral assay revealed an EC50 of 113.92 ± 4.58 μg/ml against HAV, resulting in a selectivity index (SI) of 1.92. This SI indicates limited selectivity compared to the reference drug amantadine, which exhibited an EC50 of 5.67 ± 0.71 μg/ml and an SI of 53.41.
DISCUSSION: The recombinant expression of shepherin II was successfully achieved and confirmed by mass spectrometry and SDS-PAGE. The peptide showed measurable antiviral activity against HAV.
CONCLUSION: This study demonstrated the feasibility of recombinant shepherin II production and assessed its antiviral activity. However, the limited selectivity index of shepherin II remains a challenge that needs to be addressed through molecular modification or alternative delivery strategies to improve its clinical potential.