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首页> 外文期刊>Protein Expression and Purification >Expression and assembly of Arabidopsis thaliana pyruvate dehydrogenase in insect cell cytoplasm
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Expression and assembly of Arabidopsis thaliana pyruvate dehydrogenase in insect cell cytoplasm

机译：拟南芥丙酮酸脱氢酶在昆虫细胞质中的表达和装配

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A vector was constructed for expression of Arabidopsis thaliana mitochondrial pyruvate dehydrogenase (E1) in the cytoplasm of Trichoplusia ni cells. The construct, pDDR101, comprises the mature-E1alpha coding sequence under control of the Polh promoter, plus the mature-E1beta coding sequence under control of the p10 promoter. The E1alpha sequence was engineered to include a N-terminal His-tag. When protein samples were subjected to immobilized metal ion affinity chromatography, the alpha- and beta-subunits co-eluted, indicating association. When the recombinant protein sample was further analyzed by gel permeation chromatography, it was demonstrated that a significant amount eluted at a size consistent with assembly into an alpha2beta2 heterotetramer. Recombinant E1 was able to decarboxylate [1-C-14]pyruvate, and was a substrate for in vitro phosphorylation by El-kinase. (C) 2002 Elsevier Science (USA). All rights reserved. [References: 27]
机译：构建了一种载体，用于在拟南芥ni细胞的细胞质中表达拟南芥线粒体丙酮酸脱氢酶（E1）。构建体pDDR101包含在Pohl启动子控制下的成熟E1alpha编码序列，以及在p10启动子控制下的成熟E1beta编码序列。 E1alpha序列经过工程改造，以包含N端His-tag。对蛋白质样品进行固定化的金属离子亲和层析后，α和β亚基共洗脱，表明缔合。当通过凝胶渗透色谱法进一步分析重组蛋白样品时，证明了以与组装成α2β2异四聚体一致的大小洗脱了显着量。重组E1能够使[1-C-14]丙酮酸脱羧，并且是通过El激酶进行体外磷酸化的底物。 （C）2002 Elsevier Science（美国）。版权所有。 [参考：27]

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