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BMC Plant Biology

, 5:19

First Online: 25 September 2005Received: 27 May 2005Accepted: 25 September 2005

Abstract

BackgroundThe 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.

ResultsTwo 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 Kmof 0.024 μM and a Vmaxof 4.4 pkat-mg toward 1β,2β,3β-H3GA20. The GA 3-oxidase from Arabidopsis thaliana, AtGA3OX4, was expressed as a polyHis-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 Kmof 0.82 μM and Vmaxof 52,500 pkat-mg toward 1β,2β,3β-H3GA20.

ConclusionFusion 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.

List of abbreviationsGA– gibberellin

GST– glutathione-s-transferase

IMAC– immobilised metal affinity chromatography

kat– katal

β-ME –β-mercaptoethanol

2ODD– 2-oxoglutarate-dependent dioxygenase

PBS– phosphate-buffered saline

PCR– polymerase chain reaction

PMSF– phenylmethylsulfonyl fluoride

TRX– thioredoxin

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2229-5-19 contains supplementary material, which is available to authorized users.

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Autor: Diane R Lester - Andy Phillips - Peter Hedden - Inger Andersson

Fuente: https://link.springer.com/







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