Title:Expression and Purification of His-Tagged Variants of Human Hepatitis A
Virus 3C Protease
Volume: 31
Issue: 4
Author(s): Maria A. Karaseva, Vladislav A. Gramma, Dina R. Safina, Natalia A. Lunina, Alexey A. Komissarov, Sergey V. Kostrov and Ilya V. Demidyuk*
Affiliation:
- National Research Centre "Kurchatov Institute", Moscow 123182, Russia
Keywords:
Hepatitis A virus 3C protease, immobilized metal ion affinity chromatography, polyhistidine-tagged protein, recombinant protein expression, proteolytic activity assay, chromogenic substrate.
Abstract:
Background: Protease 3C (3Cpro) is the only protease encoded in the human hepatitis
A virus genome and is considered as 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.
Objective: 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.
Methods: 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.
Results: 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.
Conclusion: We demonstrated the applicability of our approach for producing recombinant, enzymatically
active 3Cpro.
