Structural modeling, expression and purification of Chimeric chitinase 42 containing His-tag in Nicotiana tabacum hairy root system
Chimeric chitinase42 (Chit42 containing ChBD) has great potential as a candidate for digesting and recycling chitin as a beneficial nutrient, which can be produced in bioreactors. The plant is one of the most efficient bioreactors that can produce the eukaryotic proteins in active forms. With the pl...
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| Format: | Article |
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University of Belgrade - Faculty of Agriculture, Belgrade
2023-01-01
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| Series: | Journal of Agricultural Sciences (Belgrade) |
| Subjects: | |
| Online Access: | https://scindeks-clanci.ceon.rs/data/pdf/1450-8109/2023/1450-81092304473S.pdf |
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| author | Soleimani Faranak Motallebi Mostafa Zamani Mohammadreza Jourabchi Esmat |
| author_facet | Soleimani Faranak Motallebi Mostafa Zamani Mohammadreza Jourabchi Esmat |
| author_sort | Soleimani Faranak |
| collection | DOAJ |
| description | Chimeric chitinase42 (Chit42 containing ChBD) has great potential as a candidate for digesting and recycling chitin as a beneficial nutrient, which can be produced in bioreactors. The plant is one of the most efficient bioreactors that can produce the eukaryotic proteins in active forms. With the plant hairy root system, it is possible to express a variety of recombinant proteins cost-effectively, easily, and quickly. Due to the huge amount of proteins in plants, protein purification can be facilitated by the use of the His-tag. In this research, different computer programs were used for the three-dimensional structural analysis of Chimeric chitinase42 containing His-tag. The results showed that these comparative modeling approaches had a remarkable degree of accuracy in predicting the fused protein structure. The Z-score of -9.38 and -3.64 predicted for Chit42 and ChBD by ProSA represents the good quality of the model. In addition, bioinformatic observations showed that the His-tag was exposed and can be used to purify the Chimeric chitinase42. The Chimeric chitinase42 containing a His-tag was expressed in Nicotiana tabacum hairy roots, and the role of the His-tag in the detection by Western blot and purification using a Ni-NTA column was investigated. The presence of the Chimeric chitinase42 was confirmed by analyzing root extracts using SDS-PAGE and Western blot. The purification step was achieved using the His-tag and the Ni-NTA column. The plant-derived Chimeric chitinase42 was confirmed to be biologically active by measuring the chitinase activity of the purified protein on a media plate containing colloidal chitin. |
| first_indexed | 2024-03-08T04:10:42Z |
| format | Article |
| id | doaj.art-9f08dc716cd84d50869b8d42c43fe22f |
| institution | Directory Open Access Journal |
| issn | 1450-8109 2406-0968 |
| language | English |
| last_indexed | 2024-03-08T04:10:42Z |
| publishDate | 2023-01-01 |
| publisher | University of Belgrade - Faculty of Agriculture, Belgrade |
| record_format | Article |
| series | Journal of Agricultural Sciences (Belgrade) |
| spelling | doaj.art-9f08dc716cd84d50869b8d42c43fe22f2024-02-08T22:36:52ZengUniversity of Belgrade - Faculty of Agriculture, BelgradeJournal of Agricultural Sciences (Belgrade)1450-81092406-09682023-01-0168447348710.2298/JAS2304473S1450-81092304473SStructural modeling, expression and purification of Chimeric chitinase 42 containing His-tag in Nicotiana tabacum hairy root systemSoleimani Faranak0Motallebi Mostafa1Zamani Mohammadreza2Jourabchi Esmat3National Institute of Genetic Engineering and Biotechnology (NIGEB), Plant Molecular Biotechnology Department, Tehran, IranNational Institute of Genetic Engineering and Biotechnology (NIGEB), Plant Molecular Biotechnology Department, Tehran, IranNational Institute of Genetic Engineering and Biotechnology (NIGEB), Plant Molecular Biotechnology Department, Tehran, IranNational Institute of Genetic Engineering and Biotechnology (NIGEB), Plant Molecular Biotechnology Department, Tehran, IranChimeric chitinase42 (Chit42 containing ChBD) has great potential as a candidate for digesting and recycling chitin as a beneficial nutrient, which can be produced in bioreactors. The plant is one of the most efficient bioreactors that can produce the eukaryotic proteins in active forms. With the plant hairy root system, it is possible to express a variety of recombinant proteins cost-effectively, easily, and quickly. Due to the huge amount of proteins in plants, protein purification can be facilitated by the use of the His-tag. In this research, different computer programs were used for the three-dimensional structural analysis of Chimeric chitinase42 containing His-tag. The results showed that these comparative modeling approaches had a remarkable degree of accuracy in predicting the fused protein structure. The Z-score of -9.38 and -3.64 predicted for Chit42 and ChBD by ProSA represents the good quality of the model. In addition, bioinformatic observations showed that the His-tag was exposed and can be used to purify the Chimeric chitinase42. The Chimeric chitinase42 containing a His-tag was expressed in Nicotiana tabacum hairy roots, and the role of the His-tag in the detection by Western blot and purification using a Ni-NTA column was investigated. The presence of the Chimeric chitinase42 was confirmed by analyzing root extracts using SDS-PAGE and Western blot. The purification step was achieved using the His-tag and the Ni-NTA column. The plant-derived Chimeric chitinase42 was confirmed to be biologically active by measuring the chitinase activity of the purified protein on a media plate containing colloidal chitin.https://scindeks-clanci.ceon.rs/data/pdf/1450-8109/2023/1450-81092304473S.pdfchimeric chitinase 42his-tagtobacco hairy rootstructural modeling |
| spellingShingle | Soleimani Faranak Motallebi Mostafa Zamani Mohammadreza Jourabchi Esmat Structural modeling, expression and purification of Chimeric chitinase 42 containing His-tag in Nicotiana tabacum hairy root system Journal of Agricultural Sciences (Belgrade) chimeric chitinase 42 his-tag tobacco hairy root structural modeling |
| title | Structural modeling, expression and purification of Chimeric chitinase 42 containing His-tag in Nicotiana tabacum hairy root system |
| title_full | Structural modeling, expression and purification of Chimeric chitinase 42 containing His-tag in Nicotiana tabacum hairy root system |
| title_fullStr | Structural modeling, expression and purification of Chimeric chitinase 42 containing His-tag in Nicotiana tabacum hairy root system |
| title_full_unstemmed | Structural modeling, expression and purification of Chimeric chitinase 42 containing His-tag in Nicotiana tabacum hairy root system |
| title_short | Structural modeling, expression and purification of Chimeric chitinase 42 containing His-tag in Nicotiana tabacum hairy root system |
| title_sort | structural modeling expression and purification of chimeric chitinase 42 containing his tag in nicotiana tabacum hairy root system |
| topic | chimeric chitinase 42 his-tag tobacco hairy root structural modeling |
| url | https://scindeks-clanci.ceon.rs/data/pdf/1450-8109/2023/1450-81092304473S.pdf |
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