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Protein & Peptide Letters

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ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

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Research Article

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

Author(s): Maria A. Karaseva, Vladislav A. Gramma, Dina R. Safina, Natalia A. Lunina, Alexey A. Komissarov, Sergey V. Kostrov and Ilya V. Demidyuk*

Volume 31, Issue 4, 2024

Published on: 19 April, 2024

Page: [305 - 311] Pages: 7

DOI: 10.2174/0109298665293548240327082821

Price: $65

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.

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[1]
Sun, D.; Chen, S.; Cheng, A.; Wang, M. Roles of the picornaviral 3C proteinase in the viral life cycle and host cells. Viruses, 2016, 8(3), 82.
[http://dx.doi.org/10.3390/v8030082] [PMID: 26999188]
[2]
McKnight, K.L.; Lemon, S.M.; Hepatitis, A. Hepatitis A virus genome organization and replication strategy. Cold Spring Harb. Perspect. Med., 2018, 8(12), a033480.
[http://dx.doi.org/10.1101/cshperspect.a033480] [PMID: 29610147]
[3]
Shubin, A.V.; Demidyuk, I.V.; Lunina, N.A.; Komissarov, A.A.; Roschina, M.P.; Leonova, O.G.; Kostrov, S.V. Protease 3C of hepatitis A virus induces vacuolization of lysosomal/endosomal organelles and caspase-independent cell death. BMC Cell Biol., 2015, 16(1), 4.
[http://dx.doi.org/10.1186/s12860-015-0050-z] [PMID: 25886889]
[4]
Shubin, A.V.; Lunina, N.A.; Shedova, E.N.; Roshina, M.P.; Demidyuk, I.V.; Vinogradova, T.V.; Kopantsev, E.P.; Chernov, I.P.; Kostrov, S.V. Evaluation of the toxic effects evoked by the transient expression of protease genes from human pathogens in HEK293 cells. Appl. Biochem. Microbiol., 2013, 49(9), 750-755.
[http://dx.doi.org/10.1134/S0003683813090044]
[5]
Komissarov, A.A.; Karaseva, M.A.; Roschina, M.P.; Shubin, A.V.; Lunina, N.A.; Kostrov, S.V.; Demidyuk, I.V. Individual expression of hepatitis A Virus 3C protease induces ferroptosis in human cells in vitro. Int. J. Mol. Sci., 2021, 22(15), 7906.
[http://dx.doi.org/10.3390/ijms22157906] [PMID: 34360671]
[6]
Selina, P.I.; Karaseva, M.A.; Komissarov, A.A.; Safina, D.R.; Lunina, N.A.; Roschina, M.P.; Sverdlov, E.D.; Demidyuk, I.V.; Kostrov, S.V. Embryotoxic activity of 3C protease of human hepatitis A virus in developing Danio rerio embryos. Sci. Rep., 2021, 11(1), 18196.
[http://dx.doi.org/10.1038/s41598-021-97641-5] [PMID: 34521911]
[7]
Shubin, A.V.; Komissarov, A.A.; Karaseva, M.A.; Padman, B.S.; Kostrov, S.V.; Demidyuk, I.V. Human hepatitis A virus 3C protease exerts a cytostatic effect on Saccharomyces cerevisiae and affects the vacuolar compartment. Biologia, 2021, 76(1), 321-327.
[http://dx.doi.org/10.2478/s11756-020-00569-w]
[8]
Feng, H.; Stockwell, B.R. Unsolved mysteries: How does lipid peroxidation cause ferroptosis? PLoS Biol., 2018, 16(5), e2006203.
[http://dx.doi.org/10.1371/journal.pbio.2006203] [PMID: 29795546]
[9]
Komissarov, A.; Demidyuk, I.; Safina, D.; Roschina, M.; Shubin, A.; Lunina, N.; Karaseva, M.; Kostrov, S. Cytotoxic effect of co-expression of human hepatitis A virus 3C protease and bifunctional suicide protein FCU1 genes in a bicistronic vector. Mol. Biol. Rep., 2017, 44(4), 323-332.
[http://dx.doi.org/10.1007/s11033-017-4113-4] [PMID: 28748410]
[10]
Komissarov, A.A.; Kostrov, S.V.; Demidyuk, I.V. In vitro assay for the evaluation of cytotoxic effects provided by a combination of suicide and killer genes in a bicistronic vector. Methods Mol. Biol., 2019, 1895, 135-147.
[http://dx.doi.org/10.1007/978-1-4939-8922-5_11] [PMID: 30539535]
[11]
Li, J.; Cao, F.; Yin, H.; Huang, Z.; Lin, Z.; Mao, N.; Sun, B.; Wang, G. Ferroptosis: Past, present and future. Cell Death Dis., 2020, 11(2), 88.
[http://dx.doi.org/10.1038/s41419-020-2298-2] [PMID: 32015325]
[12]
Malcolm, B.A.; Chin, S.M.; Jewell, D.A.; Stratton-Thomas, J.R.; Thudium, K.B.; Ralston, R.; Rosenberg, S. Expression and characterization of recombinant hepatitis A virus 3C proteinase. Biochemistry, 1992, 31(13), 3358-3363.
[http://dx.doi.org/10.1021/bi00128a008] [PMID: 1313294]
[13]
Cui, S.; Wang, J.; Fan, T.; Qin, B.; Guo, L.; Lei, X.; Wang, J.; Wang, M.; Jin, Q. Crystal structure of human enterovirus 71 3C protease. J. Mol. Biol., 2011, 408(3), 449-461.
[http://dx.doi.org/10.1016/j.jmb.2011.03.007] [PMID: 21396941]
[14]
Rozovics, J.M.; Chase, A.J.; Cathcart, A.L.; Chou, W.; Gershon, P.D.; Palusa, S.; Wilusz, J.; Semler, B.L. Picornavirus modification of a host mRNA decay protein. MBio, 2012, 3(6), e00431-12.
[http://dx.doi.org/10.1128/mBio.00431-12] [PMID: 23131833]
[15]
Jagdeo, J.M.; Dufour, A.; Fung, G.; Luo, H.; Kleifeld, O.; Overall, C.M.; Jan, E. Heterogeneous nuclear ribonucleoprotein m facilitates enterovirus infection. J. Virol., 2015, 89(14), 7064-7078.
[http://dx.doi.org/10.1128/JVI.02977-14] [PMID: 25926642]
[16]
Sun, D.; Wang, M.; Wen, X.; Mao, S.; Cheng, A.; Jia, R.; Yang, Q.; Wu, Y.; Zhu, D.; Chen, S.; Liu, M.; Zhao, X.; Zhang, S.; Chen, X.; Liu, Y.; Yu, Y.; Zhang, L. Biochemical characterization of recombinant avihepatovirus 3C protease and its localization. Virol. J., 2019, 16(1), 54.
[http://dx.doi.org/10.1186/s12985-019-1155-3] [PMID: 31036013]
[17]
Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227(5259), 680-685.
[http://dx.doi.org/10.1038/227680a0] [PMID: 5432063]
[18]
Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 1976, 72(1-2), 248-254.
[http://dx.doi.org/10.1016/0003-2697(76)90527-3] [PMID: 942051]
[19]
Gasparov, V.S.; Degtiar’, V.G. Protein determination by binding with the dye Coomassie brilliant blue G-250. Biokhimiia, 1994, 59(6), 763-777.
[PMID: 7521220]
[20]
Peters, H.; Kusov, Y.Y.; Meyer, S.; Benie, A.J.; Bäuml, E.; Wolff, M.; Rademacher, C.; Peters, T.; Gauss-Müller, V. Hepatitis A virus proteinase 3C binding to viral RNA: correlation with substrate binding and enzyme dimerization. Biochem. J., 2005, 385(2), 363-370.
[http://dx.doi.org/10.1042/BJ20041153] [PMID: 15361063]
[21]
Schechter, I.; Berger, A. On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Commun., 1967, 27(2), 157-162.
[http://dx.doi.org/10.1016/S0006-291X(67)80055-X] [PMID: 6035483]
[22]
Jewell, D.A.; Swietnicki, W.; Dunn, B.M.; Malcolm, B.A. Hepatitis A virus 3C proteinase substrate specificity. Biochemistry, 1992, 31(34), 7862-7869.
[http://dx.doi.org/10.1021/bi00149a017] [PMID: 1510973]
[23]
Chernaia, M.M.; Malcolm, B.A.; Allaire, M.; James, M.N.G. Hepatitis A virus 3C proteinase: Some properties, crystallization and preliminary crystallographic characterization. J. Mol. Biol., 1993, 234(3), 890-893.
[http://dx.doi.org/10.1006/jmbi.1993.1636] [PMID: 8254682]
[24]
Kusov, Y.Y.; Gauss-Müller, V. In vitro RNA binding of the hepatitis A virus proteinase 3C (HAV 3Cpro) to secondary structure elements within the 5′ terminus of the HAV genome. RNA, 1997, 3(3), 291-302.
[PMID: 9056766]
[25]
Mosimann, S.C.; Cherney, M.M.; Sia, S.; Plotch, S.; James, M.N.G. Refined X-ray crystallographic structure of the poliovirus 3C gene product 1 1Edited By D. Rees. J. Mol. Biol., 1997, 273(5), 1032-1047.
[http://dx.doi.org/10.1006/jmbi.1997.1306] [PMID: 9367789]
[26]
Schultheiss, T.; Sommergruber, W.; Kusov, Y.; Gauss-Müller, V. Cleavage specificity of purified recombinant hepatitis A virus 3C proteinase on natural substrates. J. Virol., 1995, 69(3), 1727-1733.
[http://dx.doi.org/10.1128/jvi.69.3.1727-1733.1995] [PMID: 7853510]

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