Advancing large-scale production of TEV protease through an innovative NT* tag-based fusion construct
Tobacco etch virus Protease (TEVp), a cysteine protease, is renowned for its remarkable specific proteolysis, making it an invaluable tool for removing fusion tags from recombinant proteins. However, TEV protease's inherent insolubility limits its broad application. Fusion constructs like an N-...
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| Format: | Article |
| Language: | English |
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Elsevier
2023-01-01
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| Series: | Current Research in Structural Biology |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2665928X23000120 |
| _version_ | 1797397562238435328 |
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| author | Pragyan P. Parida Deepa Saraswathi Subbarao M.V. Mopidevi Sreejith Raran-Kurussi |
| author_facet | Pragyan P. Parida Deepa Saraswathi Subbarao M.V. Mopidevi Sreejith Raran-Kurussi |
| author_sort | Pragyan P. Parida |
| collection | DOAJ |
| description | Tobacco etch virus Protease (TEVp), a cysteine protease, is renowned for its remarkable specific proteolysis, making it an invaluable tool for removing fusion tags from recombinant proteins. However, TEV protease's inherent insolubility limits its broad application. Fusion constructs like an N-terminal MBP fusion, known for its improved solubility, have been employed for TEVp production to address this issue. In this study, we fused the TEVp with the N-terminal domain of the spider silk protein, specifically utilizing a charge-reversed mutant (D40K/K65D) of the N-terminal domain of major ampullate spidroin-1 protein from Euprosthenops australis, referred to as NT*. This fusion construct contains a TEVp cleavage site, enabling intracellular self-processing and the release of a His7-tagged protease. The significant increase in soluble protein expression allowed us to purify approximately 90–100 mg of TEVp from a 1-L E. coli culture, surpassing previous findings by a considerable margin. The enzyme remained stable and catalytically active even after several months of storage in a deep freezer (−80 °C). |
| first_indexed | 2024-03-09T01:11:51Z |
| format | Article |
| id | doaj.art-9ce07f7fc920455db4b9de09e55e4898 |
| institution | Directory Open Access Journal |
| issn | 2665-928X |
| language | English |
| last_indexed | 2024-03-09T01:11:51Z |
| publishDate | 2023-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Current Research in Structural Biology |
| spelling | doaj.art-9ce07f7fc920455db4b9de09e55e48982023-12-11T04:17:03ZengElsevierCurrent Research in Structural Biology2665-928X2023-01-016100106Advancing large-scale production of TEV protease through an innovative NT* tag-based fusion constructPragyan P. Parida0Deepa Saraswathi1Subbarao M.V. Mopidevi2Sreejith Raran-Kurussi3Tata Institute of Fundamental Research Hyderabad, 36/P Gopanpally Village, Ranga Reddy District, Serilingampally, Hyderabad, 500046, Telangana, IndiaTata Institute of Fundamental Research Hyderabad, 36/P Gopanpally Village, Ranga Reddy District, Serilingampally, Hyderabad, 500046, Telangana, IndiaTata Institute of Fundamental Research Hyderabad, 36/P Gopanpally Village, Ranga Reddy District, Serilingampally, Hyderabad, 500046, Telangana, IndiaCorresponding author. Tata Institute of Fundamental Research, 36/P Gopanpally Village, Ranga Reddy District, Serilingampally Hyderabad, 500107, Telangana, India.; Tata Institute of Fundamental Research Hyderabad, 36/P Gopanpally Village, Ranga Reddy District, Serilingampally, Hyderabad, 500046, Telangana, IndiaTobacco etch virus Protease (TEVp), a cysteine protease, is renowned for its remarkable specific proteolysis, making it an invaluable tool for removing fusion tags from recombinant proteins. However, TEV protease's inherent insolubility limits its broad application. Fusion constructs like an N-terminal MBP fusion, known for its improved solubility, have been employed for TEVp production to address this issue. In this study, we fused the TEVp with the N-terminal domain of the spider silk protein, specifically utilizing a charge-reversed mutant (D40K/K65D) of the N-terminal domain of major ampullate spidroin-1 protein from Euprosthenops australis, referred to as NT*. This fusion construct contains a TEVp cleavage site, enabling intracellular self-processing and the release of a His7-tagged protease. The significant increase in soluble protein expression allowed us to purify approximately 90–100 mg of TEVp from a 1-L E. coli culture, surpassing previous findings by a considerable margin. The enzyme remained stable and catalytically active even after several months of storage in a deep freezer (−80 °C).http://www.sciencedirect.com/science/article/pii/S2665928X23000120NT*Fusion proteinTEV proteaseCleavage sitePurificationEnzymatic activity |
| spellingShingle | Pragyan P. Parida Deepa Saraswathi Subbarao M.V. Mopidevi Sreejith Raran-Kurussi Advancing large-scale production of TEV protease through an innovative NT* tag-based fusion construct Current Research in Structural Biology NT* Fusion protein TEV protease Cleavage site Purification Enzymatic activity |
| title | Advancing large-scale production of TEV protease through an innovative NT* tag-based fusion construct |
| title_full | Advancing large-scale production of TEV protease through an innovative NT* tag-based fusion construct |
| title_fullStr | Advancing large-scale production of TEV protease through an innovative NT* tag-based fusion construct |
| title_full_unstemmed | Advancing large-scale production of TEV protease through an innovative NT* tag-based fusion construct |
| title_short | Advancing large-scale production of TEV protease through an innovative NT* tag-based fusion construct |
| title_sort | advancing large scale production of tev protease through an innovative nt tag based fusion construct |
| topic | NT* Fusion protein TEV protease Cleavage site Purification Enzymatic activity |
| url | http://www.sciencedirect.com/science/article/pii/S2665928X23000120 |
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