The main protease 3CLpro of the SARS-CoV-2 virus: how to turn an enemy into a helper
Despite the long history of use and the knowledge of the genetics and biochemistry of E. coli , problems are still possible in obtaining a soluble form of recombinant proteins in this system. Although, soluble protein can be obtained both in the cytoplasm and in the periplasm of the bacterial cell....
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| Published in | Frontiers in bioengineering and biotechnology Vol. 11; p. 1187761 |
|---|---|
| Main Authors | , , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Switzerland
Frontiers Media S.A
29.06.2023
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| Abstract | Despite the long history of use and the knowledge of the genetics and biochemistry of
E. coli
, problems are still possible in obtaining a soluble form of recombinant proteins in this system. Although, soluble protein can be obtained both in the cytoplasm and in the periplasm of the bacterial cell. The latter is a priority strategy for obtaining soluble proteins. The fusion protein technology followed by detachment of the fusion protein with proteases is used to transfer the target protein into the periplasmic space of
E. coli
. We have continued for the first time to use the main viral protease 3CL of the SARS-CoV-2 virus for this purpose. We obtained a recombinant 3CL protease and studied its complex catalytic properties. The authenticity of the resulting recombinant enzyme, were confirmed by specific activity analysis and activity suppression by the known low-molecular-weight inhibitors. The catalytic efficiency of 3CL (0.17 ± 0.02 µM-1-s-1) was shown to be one order of magnitude higher than that of the widely used tobacco etch virus protease (0.013 ± 0.003 µM-1-s-1). The application of the 3CL gene in genetically engineered constructs provided efficient specific proteolysis of fusion proteins, which we demonstrated using the receptor-binding domain of SARS-CoV-2 spike protein and GST fusion protein. The solubility and immunochemical properties of RBD were preserved. It is very important that in work we have shown that 3CL protease works effectively directly in
E. coli
cells when co-expressed with the target fusion protein, as well as when expressed as part of a chimeric protein containing the target protein, fusion partner, and 3CL itself. The results obtained in the work allow expanding the repertoire of specific proteases for researchers and biotechnologists. |
|---|---|
| AbstractList | Despite the long history of use and the knowledge of the genetics and biochemistry of
E. coli
, problems are still possible in obtaining a soluble form of recombinant proteins in this system. Although, soluble protein can be obtained both in the cytoplasm and in the periplasm of the bacterial cell. The latter is a priority strategy for obtaining soluble proteins. The fusion protein technology followed by detachment of the fusion protein with proteases is used to transfer the target protein into the periplasmic space of
E. coli
. We have continued for the first time to use the main viral protease 3CL of the SARS-CoV-2 virus for this purpose. We obtained a recombinant 3CL protease and studied its complex catalytic properties. The authenticity of the resulting recombinant enzyme, were confirmed by specific activity analysis and activity suppression by the known low-molecular-weight inhibitors. The catalytic efficiency of 3CL (0.17 ± 0.02 µM-1-s-1) was shown to be one order of magnitude higher than that of the widely used tobacco etch virus protease (0.013 ± 0.003 µM-1-s-1). The application of the 3CL gene in genetically engineered constructs provided efficient specific proteolysis of fusion proteins, which we demonstrated using the receptor-binding domain of SARS-CoV-2 spike protein and GST fusion protein. The solubility and immunochemical properties of RBD were preserved. It is very important that in work we have shown that 3CL protease works effectively directly in
E. coli
cells when co-expressed with the target fusion protein, as well as when expressed as part of a chimeric protein containing the target protein, fusion partner, and 3CL itself. The results obtained in the work allow expanding the repertoire of specific proteases for researchers and biotechnologists. Despite the long history of use and the knowledge of the genetics and biochemistry of , problems are still possible in obtaining a soluble form of recombinant proteins in this system. Although, soluble protein can be obtained both in the cytoplasm and in the periplasm of the bacterial cell. The latter is a priority strategy for obtaining soluble proteins. The fusion protein technology followed by detachment of the fusion protein with proteases is used to transfer the target protein into the periplasmic space of . We have continued for the first time to use the main viral protease 3CL of the SARS-CoV-2 virus for this purpose. We obtained a recombinant 3CL protease and studied its complex catalytic properties. The authenticity of the resulting recombinant enzyme, were confirmed by specific activity analysis and activity suppression by the known low-molecular-weight inhibitors. The catalytic efficiency of 3CL (0.17 ± 0.02 µM-1-s-1) was shown to be one order of magnitude higher than that of the widely used tobacco etch virus protease (0.013 ± 0.003 µM-1-s-1). The application of the 3CL gene in genetically engineered constructs provided efficient specific proteolysis of fusion proteins, which we demonstrated using the receptor-binding domain of SARS-CoV-2 spike protein and GST fusion protein. The solubility and immunochemical properties of RBD were preserved. It is very important that in work we have shown that 3CL protease works effectively directly in cells when co-expressed with the target fusion protein, as well as when expressed as part of a chimeric protein containing the target protein, fusion partner, and 3CL itself. The results obtained in the work allow expanding the repertoire of specific proteases for researchers and biotechnologists. Despite the long history of use and the knowledge of the genetics and biochemistry of E. coli, problems are still possible in obtaining a soluble form of recombinant proteins in this system. Although, soluble protein can be obtained both in the cytoplasm and in the periplasm of the bacterial cell. The latter is a priority strategy for obtaining soluble proteins. The fusion protein technology followed by detachment of the fusion protein with proteases is used to transfer the target protein into the periplasmic space of E. coli. We have continued for the first time to use the main viral protease 3CL of the SARS-CoV-2 virus for this purpose. We obtained a recombinant 3CL protease and studied its complex catalytic properties. The authenticity of the resulting recombinant enzyme, were confirmed by specific activity analysis and activity suppression by the known low-molecular-weight inhibitors. The catalytic efficiency of 3CL (0.17 ± 0.02 µM-1-s-1) was shown to be one order of magnitude higher than that of the widely used tobacco etch virus protease (0.013 ± 0.003 µM-1-s-1). The application of the 3CL gene in genetically engineered constructs provided efficient specific proteolysis of fusion proteins, which we demonstrated using the receptor-binding domain of SARS-CoV-2 spike protein and GST fusion protein. The solubility and immunochemical properties of RBD were preserved. It is very important that in work we have shown that 3CL protease works effectively directly in E. coli cells when co-expressed with the target fusion protein, as well as when expressed as part of a chimeric protein containing the target protein, fusion partner, and 3CL itself. The results obtained in the work allow expanding the repertoire of specific proteases for researchers and biotechnologists.Despite the long history of use and the knowledge of the genetics and biochemistry of E. coli, problems are still possible in obtaining a soluble form of recombinant proteins in this system. Although, soluble protein can be obtained both in the cytoplasm and in the periplasm of the bacterial cell. The latter is a priority strategy for obtaining soluble proteins. The fusion protein technology followed by detachment of the fusion protein with proteases is used to transfer the target protein into the periplasmic space of E. coli. We have continued for the first time to use the main viral protease 3CL of the SARS-CoV-2 virus for this purpose. We obtained a recombinant 3CL protease and studied its complex catalytic properties. The authenticity of the resulting recombinant enzyme, were confirmed by specific activity analysis and activity suppression by the known low-molecular-weight inhibitors. The catalytic efficiency of 3CL (0.17 ± 0.02 µM-1-s-1) was shown to be one order of magnitude higher than that of the widely used tobacco etch virus protease (0.013 ± 0.003 µM-1-s-1). The application of the 3CL gene in genetically engineered constructs provided efficient specific proteolysis of fusion proteins, which we demonstrated using the receptor-binding domain of SARS-CoV-2 spike protein and GST fusion protein. The solubility and immunochemical properties of RBD were preserved. It is very important that in work we have shown that 3CL protease works effectively directly in E. coli cells when co-expressed with the target fusion protein, as well as when expressed as part of a chimeric protein containing the target protein, fusion partner, and 3CL itself. The results obtained in the work allow expanding the repertoire of specific proteases for researchers and biotechnologists. Despite the long history of use and the knowledge of the genetics and biochemistry of E. coli, problems are still possible in obtaining a soluble form of recombinant proteins in this system. Although, soluble protein can be obtained both in the cytoplasm and in the periplasm of the bacterial cell. The latter is a priority strategy for obtaining soluble proteins. The fusion protein technology followed by detachment of the fusion protein with proteases is used to transfer the target protein into the periplasmic space of E. coli. We have continued for the first time to use the main viral protease 3CL of the SARS-CoV-2 virus for this purpose. We obtained a recombinant 3CL protease and studied its complex catalytic properties. The authenticity of the resulting recombinant enzyme, were confirmed by specific activity analysis and activity suppression by the known low-molecular-weight inhibitors. The catalytic efficiency of 3CL (0.17 ± 0.02 µM-1-s-1) was shown to be one order of magnitude higher than that of the widely used tobacco etch virus protease (0.013 ± 0.003 µM-1-s-1). The application of the 3CL gene in genetically engineered constructs provided efficient specific proteolysis of fusion proteins, which we demonstrated using the receptor-binding domain of SARS-CoV-2 spike protein and GST fusion protein. The solubility and immunochemical properties of RBD were preserved. It is very important that in work we have shown that 3CL protease works effectively directly in E. coli cells when co-expressed with the target fusion protein, as well as when expressed as part of a chimeric protein containing the target protein, fusion partner, and 3CL itself. The results obtained in the work allow expanding the repertoire of specific proteases for researchers and biotechnologists. |
| Author | Belenkaya, Svetlana V. Chirkova, Varvara Yu Shanshin, Daniil V. Volosnikova, Ekaterina A. Merkuleva, Iuliia A. Sharlaeva, Elena A. Vatsadze, Sergey Z. Khvostov, Mikhail V. Shcherbakov, Dmitriy N. Salakhutdinov, Nariman F. Yarovaya, Olga I. |
| AuthorAffiliation | 5 N.D Zelinsky Institute of Organic Chemistry , Russian Academy of Sciences , Moscow , Russia 1 Laboratory of Bionanotechnology, Microbiology and Virology, Novosibirsk State University , Novosibirsk , Russia 2 State Research Center of Virology and Biotechnology VECTOR , Koltsovo , Russia 4 Department of Physical-Chemistry Biology and Biotechnology , Altay State University , Barnaul , Russia 3 Department of Medicinal Chemistry , N.N Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS , Novosibirsk , Russia |
| AuthorAffiliation_xml | – name: 1 Laboratory of Bionanotechnology, Microbiology and Virology, Novosibirsk State University , Novosibirsk , Russia – name: 2 State Research Center of Virology and Biotechnology VECTOR , Koltsovo , Russia – name: 5 N.D Zelinsky Institute of Organic Chemistry , Russian Academy of Sciences , Moscow , Russia – name: 3 Department of Medicinal Chemistry , N.N Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS , Novosibirsk , Russia – name: 4 Department of Physical-Chemistry Biology and Biotechnology , Altay State University , Barnaul , Russia |
| Author_xml | – sequence: 1 givenname: Svetlana V. surname: Belenkaya fullname: Belenkaya, Svetlana V. – sequence: 2 givenname: Iuliia A. surname: Merkuleva fullname: Merkuleva, Iuliia A. – sequence: 3 givenname: Olga I. surname: Yarovaya fullname: Yarovaya, Olga I. – sequence: 4 givenname: Varvara Yu surname: Chirkova fullname: Chirkova, Varvara Yu – sequence: 5 givenname: Elena A. surname: Sharlaeva fullname: Sharlaeva, Elena A. – sequence: 6 givenname: Daniil V. surname: Shanshin fullname: Shanshin, Daniil V. – sequence: 7 givenname: Ekaterina A. surname: Volosnikova fullname: Volosnikova, Ekaterina A. – sequence: 8 givenname: Sergey Z. surname: Vatsadze fullname: Vatsadze, Sergey Z. – sequence: 9 givenname: Mikhail V. surname: Khvostov fullname: Khvostov, Mikhail V. – sequence: 10 givenname: Nariman F. surname: Salakhutdinov fullname: Salakhutdinov, Nariman F. – sequence: 11 givenname: Dmitriy N. surname: Shcherbakov fullname: Shcherbakov, Dmitriy N. |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/37456729$$D View this record in MEDLINE/PubMed |
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| Cites_doi | 10.1006/prep.1999.1150 10.3390/v14102154 10.1006/prep.1997.0759 10.3389/fphar.2021.773198 10.1016/j.pep.2004.10.017 10.3390/molecules28010303 10.1016/S0006-291X(02)00574-0 10.3390/vaccines10010096 10.1002/pro.3668 10.1073/pnas.86.8.2540 10.1016/j.ijbiomac.2020.08.166 10.3389/fmicb.2014.00172 10.1016/j.pep.2003.08.023 10.1016/j.pep.2021.105861 10.1107/S0907444901021187 10.3390/v13020174 10.1110/ps.8.8.1668 10.1016/j.copbio.2006.06.003 10.1006/prep.2000.1251 10.1002/med.21783 10.1007/s10295-011-1082-9 10.1016/j.tibtech.2006.06.008 10.1016/j.pep.2006.10.019 10.1016/j.tibtech.2005.03.012 10.1038/s41598-020-79357-0 10.1016/0378-1119(88)90005-4 10.1002/elsc.202000106 10.1038/s41586-020-2223-y 10.4014/jmb.1412.12079 10.1016/0003-2697(81)90281-5 10.1016/j.pep.2005.03.016 10.1038/227680a0 10.1007/978-1-59745-196-3_19 10.1016/S0958-1669(00)00200-7 10.1038/s41592-019-0665-7 10.1016/0042-6822(89)90603-X 10.1002/bit.27912 10.1016/S0304-4165(00)00086-6 10.1100/tsw.2002.215 10.1016/0378-1119(88)90170-9 10.1042/bj3060589 10.1073/pnas.1601327113 10.1016/j.colsurfb.2008.09.012 10.1021/acsmedchemlett.1c00299 |
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| Copyright | Copyright © 2023 Belenkaya, Merkuleva, Yarovaya, Chirkova, Sharlaeva, Shanshin, Volosnikova, Vatsadze, Khvostov, Salakhutdinov and Shcherbakov. Copyright © 2023 Belenkaya, Merkuleva, Yarovaya, Chirkova, Sharlaeva, Shanshin, Volosnikova, Vatsadze, Khvostov, Salakhutdinov and Shcherbakov. 2023 Belenkaya, Merkuleva, Yarovaya, Chirkova, Sharlaeva, Shanshin, Volosnikova, Vatsadze, Khvostov, Salakhutdinov and Shcherbakov |
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| Keywords | 3CL protease SARS-CoV-2 RBD E. coli bacteria TEV protease |
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| License | Copyright © 2023 Belenkaya, Merkuleva, Yarovaya, Chirkova, Sharlaeva, Shanshin, Volosnikova, Vatsadze, Khvostov, Salakhutdinov and Shcherbakov. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
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E. coli
, problems are still possible in obtaining a soluble form of... Despite the long history of use and the knowledge of the genetics and biochemistry of , problems are still possible in obtaining a soluble form of recombinant... Despite the long history of use and the knowledge of the genetics and biochemistry of E. coli, problems are still possible in obtaining a soluble form of... |
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| SubjectTerms | 3CL protease Bioengineering and Biotechnology E. coli bacteria RBD SARS-CoV-2 TEV protease |
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| Title | The main protease 3CLpro of the SARS-CoV-2 virus: how to turn an enemy into a helper |
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