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首页> 外文期刊>BMC Plant Biology >Purification and kinetic studies of recombinant gibberellin dioxygenases
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Purification and kinetic studies of recombinant gibberellin dioxygenases

机译：重组赤霉素双加氧酶的纯化和动力学研究

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Background The 2-oxoglutarate-dependent dioxygenases (2ODDs) of gibberellin (GA) biosynthesis have a key role in the metabolism of a major plant hormone. The activity of recombinant GA 2ODDs from many species has been characterised in detail, however little information relates to enzyme purification. Native GA 2ODDs displayed lability during purification. Results Two GA 2ODDs were expressed in Escherichia coli and purified to homogeneity. The GA 2-oxidase from Pisum sativum L., PsGA2OX1, was expressed as a glutathione s -transferase (GST) fusion. It was purified in the three steps of affinity chromatography, GST removal and gel filtration. Highly pure PsGA2OX1 was obtained at a yield of 0.3 mg/g of cells. It displayed a K m of 0.024 μM and a V max of 4.4 pkat/mg toward [1 β ,2 β ,3 β -3H3]GA20. The GA 3-oxidase from Arabidopsis thaliana , AtGA3OX4, was expressed as a poly(His)-tagged thioredoxin fusion. It was purified by Immobilised Metal Affinity Chromatography followed by gel filtration. Cleavage of the fusion took place between the two purification steps. Highly pure AtGA3OX4 was obtained at a yield of 0.01 mg/g of cells. It displayed a K m of 0.82 μM and V max of 52,500 pkat/mg toward [1 β ,2 β ,3 β -3H3]GA20. Conclusion Fusion tags were required to stabilise and solubilise PsGA2OX1 and AtGA3OX4 during E. coli expression. The successful purification of milligram quantities of PsGA2OX1 enables mechanistic and structural studies not previously possible on GA 2ODDs. A moderate yield of pure AtGA3OX4 requires the further optimisation of the latter stages of the enzyme purification schedule. PsGA2OX1's action in planta as deduced from the effect of the null mutation sln on GA levels in seeds is in agreement with the kinetic parameters of the recombinant enzyme.
机译：背景赤霉素（GA）生物合成的2-氧戊二酸依赖性双加氧酶（2ODD）在主要植物激素的代谢中具有关键作用。已经详细描述了来自许多物种的重组GA 2ODD的活性，但是有关酶纯化的信息很少。天然GA 2ODD在纯化过程中显示出不稳定性。结果两个GA 2ODDs在大肠杆菌中表达并纯化至均质。来自豌豆（Pisum sativum L.）的GA 2-氧化酶，PsGA2OX1，被表达为谷胱甘肽S-转移酶（GST）融合体。它通过亲和色谱，GST去除和凝胶过滤三个步骤进行纯化。以0.3 mg / g细胞的产量获得了高纯度的PsGA2OX1。它显示出朝向[1β，2β，3β- 3 的K m 为0.024μM，V max 为4.4 pkat / mg。 H 3 ] GA 20 。来自拟南芥的GA 3-氧化酶AtGA3OX4被表达为聚（His）标记的硫氧还蛋白融合物。通过固定金属亲和色谱法纯化，然后进行凝胶过滤。融合物的切割在两个纯化步骤之间进行。以0.01 mg / g细胞的产量获得了高纯度的AtGA3OX4。它显示出朝向[1β，2β，3β- 3 H>的K m 为0.82μM，V max 为52,500 pkat / mg。 3 ] GA 20 。结论在大肠杆菌表达过程中需要融合标签来稳定和溶解PsGA2OX1和AtGA3OX4。毫克量的PsGA2OX1的成功纯化，可以进行以前在GA 2ODD上不可能进行的机械和结构研究。纯AtGA3OX4的中等收率需要进一步优化酶纯化程序的后期。由无效突变sln对种子中GA水平的影响推导的PsGA2OX1在植物中的作用与重组酶的动力学参数一致。

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