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| Purification and Characterization of Pyrroline-5-carboxylate Reductase from Drosophila melanogaster |
| FENG Chang-zeng1,2, XIE Yue-hui3, CAO Yi4,5, MENG Zhao-hui1 |
1. Laboratory of Molecular Cardiology, Department of Cardiology, The First Affiliated Hospital of Kunming Medical College, Kunming 650032, China;
2. The Second Department of Biological Products, Institute of Medical Biology, Chinese Academy of Medical Sciences, Kunming 650118, China;
3. Department of Computer Science, the Faculty of Basic Medicine, Kunming Medical University, Kunming 650031, China;
4. Laboratory for Conservation and Utilization of Bio-Resources and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming 650091, China;
5. Guizhou Tobacco Science Research Institute, Guiyang 550081, China |
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Abstract P5CRs exist in every domain of life, including eukarya, bacteria and archea. They catalyze the conversion from Δ1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)+ in the final step of proline biosynthesis. In order to provide the theory basis of the function and three-dimensional structure of P5CR, it was performed to research the aggregation form, enzymatic properties and optimal crystallization conditions of P5CR from Drosophila melanogaster. Total RNA from Drosophila melanogaster was isolated, and reverse transcripted into cDNA with random primers. The full-length sequence of the P5CR gene was amplified from the cDNA and comfirmed by sequencing. The coding sequence of P5CR was subcloned into Novagen pET-28a (+) vector. The target protein was over-expressed in Escherichia coli and purified to homogeneity by chromatography. The homopolymer was characterized by EGS cross-linking and size exclusion gel filtration chromatography. The thioproline dehydrogenase activity of P5CR was assayed at room temperature using a sensitive spectrophotometric method. The crystallization conditions of P5CR using hanging-drop vapor diffusion method were screened and optimized.
The main results were as follows: (1) The method of overexpression and purificaiton of Drosophila melanogaster P5CR was obtained, and the purity of P5CR is high. (2) Drosophila melanogaster P5CR mainly exists in the form of decamer in solution, on this basis, forms homopolymer. (3) P5CR can be involved in the metabolism of thioproline which was considered as a kind of anticancer drug. This enzyme has an optimal alkaline pH in this reversible reaction and is thermo stable with a half-life of approximately 15 min at 45℃. (4) One remarkable crystallization condition was obtained (0.2mol/L Ammonium phosphate dibasic, 20% PEG3350, and pH 8.0).
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Received: 23 February 2012
Published: 25 June 2012
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