Cloning, Expression and Characterization of Malate Dehydrogenase from Bamboo (Bambusa oldhamii)
| Autor: | Che-Jen Hsiao, 蕭哲仁 |
|---|---|
| Rok vydání: | 2008 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 96 Malate dehydrogenase (MDH EC 1.1.1.37), which is ubiquitous in nature, catalyzes the interconversion of oxaloacetate and malate. Higher plants contain multiple forms of MDH that differ in co-enzyme specificity, subcellular localization and physiological function. Glyoxysomal NAD-dependent MDH (gMDH) is one class of MDH that has not been extensively characterized in plants, and also there’s no any research studying the relation between gMDH and cytokinin oxidase/dehydrogenase (CKX, EC 1.55.99.12). Unexpectedly, a putative Mdh cDNA was screened with the specific probe of CKX from the cDNA library of Bambusa oldhamii in our laboratory. Here we present the cloning, characterization and prokaryotic expression of this putative Mdh. Sequence alignment shows that there’s a high homology between the deduced amino sequence of BogMDH and MDH protein in glyoxysome in Oryza sativa (96%). Nearly 57 kD fusion protein was expressed by IPTG induction in Escherichia coli BL21 (DE3), and an obvious MDH activity was detected in the protein. All these results suggest that BogMDH encodes a glyoxysomal MDH. Screened the genomic library indicated the Mdh has no intron. After preparing the polyclonal antibody by using fusion protein as the antigen, we partial purified MDH from bamboo through buffer extraction, ammonium sulfate precipitation, and Fast Protein Liquid Chromatography (FPLC). The recombinant MDH and the purified MDH both have similar characteristics in kinetics and the optimum pH. But the recombinant protein prefers a low temperature. In contrast, the purified protein favors a higher temperature. Using Superose 12 column, the molecular weight of native form bamboo MDH was estimated to be 130 ~ 115 kD, and the subunit form was about 45 kD by SDS-PAGE. It might be a homodimeric enzyme. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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