Expression and Purification of His-Tagged Variants of Human Hepatitis A Virus 3C Protease

• Published in: Protein and peptide letters
• Main Authors: Karaseva, Maria A, Gramma, Vladislav A, Safina, Dina R, Lunina, Natalia A, Komissarov, Alexey A, Kostrov, Sergey V, Demidyuk, Ilya V
• Format: Journal Article
• Language: English
• Published: Netherlands 01.01.2024
• Subjects: recombinant protein expression; proteolytic activity assay; chromogenic substrate; polyhistidine-tagged protein; Hepatitis A virus 3C protease; immobilized metal ion affinity chromatography
• ISSN: 1875-5305
• DOI: 10.2174/0109298665293548240327082821

Protease 3C (3Cpro) is the only protease encoded in the human hepatitis A virus genome and is considered a potential target for antiviral drugs due to its critical role in the viral life cycle. Additionally, 3Cpro has been identified as a potent inducer of ferroptosis, a newly described type of cell death. Therefore, studying the molecular mechanism of 3Cpro functioning can provide new insights into viral-host interaction and the biological role of ferroptosis. However, such studies require a reliable technique for producing the functionally active recombinant enzyme. Here, we expressed different modified forms of 3Cpro with a hexahistidine tag on the N- or C-terminus to investigate the applicability of Immobilized Metal Ion Affinity Chromatography (IMAC) for producing 3Cpro. We expressed the proteins in Escherichia coli and purified them using IMAC, followed by gel permeation chromatography. The enzymatic activity of the produced proteins was assayed using a specific chromogenic substrate. Our findings showed that the introduction and position of the hexahistidine tag did not affect the activity of the enzyme. However, the yield of the target protein was highest for the variant with seven C-terminal residues replaced by a hexahistidine sequence. We demonstrated the applicability of our approach for producing recombinant, enzymatically active 3Cpro.