Shepherin II Gene Synthesis and Peptide Characterization: E. coli Expression, Purification, and Antiviral Activity

Theodor Bilharz Research Institute

Bibliographic Information

Authors: Elfattah A.A.; Samir S.; Okasha H.; Atef A.A.; Taha A.

Journal: Protein and Peptide Letters

Publisher: Bentham Science Publishers

Publication Date: October 2026

Volume / Issue: Volume 32 / Issue 10

Pages: 756–768

ISSN: 9298665

DOI: 10.2174/0109298665413796251002111415

Scopus: View on Scopus

PubMed: 41140082

Document Type: Article

Authors and Affiliations

Elfattah A.A., Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, 11566, Egypt; Samir S., Department of Biochemistry and Molecular Biology, Theodor Bilharz Research Institute, Giza, 12411, Egypt; Okasha H., Department of Biochemistry and Molecular Biology, Theodor Bilharz Research Institute, Giza, 12411, Egypt; Atef A.A., Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, 11566, Egypt; Taha A., Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, 11566, Egypt

Abstract

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. 2025, Bentham Science Publishers

Keywords

antimicrobial peptide; antiviral activity; HAV; Histidine-glycine rich peptide; recombinant DNA technology; shepherin peptides; Animals; Antiviral Agents; Chlorocebus aethiops; Escherichia coli; Gene Expression; Hepatitis A virus; Peptides; Recombinant Proteins; Vero Cells; amantadine; antivirus agent; isopropyl thiogalactoside; shepherin II peptide; unclassified drug; peptide; recombinant protein; Article; bacterial gene; BL21 gene; CC50 (cytotoxic concentration); controlled study; cytotoxicity; cytotoxicity test; drug design; drug purification; drug synthesis; EC50; gene synthesis; ion exchange chromatography; mass spectrometry; molecular weight; nonhuman; peptide analysis; polyacrylamide gel electrophoresis; selectivity index; Vero cell line; animal; biosynthesis; chemistry; drug effect; genetics; isolation and purification; metabolism

Citation Information

Scopus Citations: 1

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