Molecular cloning, heterologous expression and functional characterization of gamma tocopherol methyl transferase (γ-TMT) from Glycine max
| Autor: | Kalpana Tewari, Kishwar Ali, Vaibhav Kumar, Anil Dahuja, Archana Sachdev, Sweta Kumari, Amresh Kumar |
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| Rok vydání: | 2017 |
| Předmět: |
0106 biological sciences
0301 basic medicine Molecular cloning Biology 01 natural sciences law.invention 03 medical and health sciences Gene Expression Regulation Plant law Cloning Molecular Gene Histidine chemistry.chemical_classification gamma-Tocopherol Expression vector Methyltransferases Plants Genetically Modified Molecular biology 030104 developmental biology Enzyme chemistry Biochemistry Seeds Glycine Recombinant DNA Soybeans Heterologous expression human activities 010606 plant biology & botany Biotechnology |
| Zdroj: | Protein Expression and Purification. 140:81-89 |
| ISSN: | 1046-5928 |
| Popis: | γ-Tocopherol methyltransferase (γ-TMT) (EC 2.1.1.95) is the last enzyme in the tocopherol biosynthetic pathway and it catalyzes the conversion of γ-tocopherol into α-tocopherol, the nutritionally significant and most bioactive form of vitamin E. Although the γ-TMT gene has been successfully overexpressed in many crops to enhance their α-tocopherol content but still only few attempts have been made to uncover its structural, functional and regulation aspects at protein level. In this study, we have cloned the complete 909bp coding sequence of Glycine max γ-TMT (Gm γ-TMT) gene that encodes the corresponding protein comprising of 302 amino acid residues. The deduced Gm γ-TMT protein showed 74-87% sequence identity with other characterized plant γ-TMTs. Gm γ-TMT belongs to Class I Methyl Transferases that have a Rossmann-like fold which consists of a seven-stranded β sheet joined by α helices. Heterologous expression of Gm γ-TMT in pET29a expression vector under the control of bacteriophage T7 promoter produced a 37.9 kDa recombinant Gm γ-TMT protein with histidine hexamer tag at its C-terminus. The expression of recombinant Gm γ-TMT protein was confirmed by western blotting using anti-His antibody. The recombinant protein was purified by Ni2+-NTA column chromatography. The purified protein showed SAM dependent methyltransferase activity. The α-tocopherol produced in the in-vitro reaction catalyzed by the purified enzyme was detected using reverse phase HPLC. This study has laid the foundation to unveil the biochemical understanding of Gm γ-TMT enzyme which can be further explored by studying its kinetic behaviour, substrate specificity and its interaction with other biomolecules. |
| Databáze: | OpenAIRE |
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