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首页> 外文期刊>Biotechnology Progress >Cloning and functional expression of the dps gene encoding decaprenyl diphosphate synthase from Agrobacterium tumefaciens
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Cloning and functional expression of the dps gene encoding decaprenyl diphosphate synthase from Agrobacterium tumefaciens

机译：根癌农杆菌编码癸二烯基二磷酸合酶的dps基因的克隆和功能表达

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摘要

A newly isolated gene from Agrobacterium tumefaciens (A. tumefaciens), which encoded a decaprenyl diphosphate synthase, was cloned in Escherichia coli (E. coli), and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1077 bp capable of encoding a 358-amino-acid protein with a calculated isoelectric point of pH 5.16 and a molecular mass of 38 960 Da. The primary structure of the enzyme shared significant homology with prenyl diphosphate synthases from various sources. The deduced amino acid sequence included oligopeptide DDxxD aspartate-rich domains conserved in the majority of prenyl diphosphate synthases. High levels of the active enzyme were expressed in the soluble fraction and were readily purified to homogeneity by Ni-NTA chromatography. E. coli JM109 harboring the dps gene produced ubiquinone-10 in addition to endogenous ubiquinone-8, while E. coli JM109 harboring the dps gene mutated on the DDxxD domain lost the ability to produce ubiquinone-10, which suggests that the A. tumefaciens dps gene is functionally expressed in E. coli and that it encodes a decaprenyl diphosphate synthase.
机译：从根癌农杆菌（A.tumefaciens）中新分离的基因编码了癸二烯基二磷酸合酶，被克隆到大肠杆菌（E.coli）中，并确定了其核苷酸序列。 DNA序列分析揭示了一个1077 bp的开放阅读框，它能够编码358个氨基酸的蛋白质，其等电点的计算值为pH 5.16，分子量为38960 Da。该酶的一级结构与来自各种来源的异戊二烯基二磷酸合酶具有显着的同源性。推导的氨基酸序列包括在大多数异戊二烯基二磷酸合酶中保守的富含寡肽DDxxD的天冬氨酸结构域。高水平的活性酶在可溶性级分中表达，并易于通过Ni-NTA色谱纯化至均一。携带dps基因的大肠杆菌JM109除内源性泛醌8外还产生了泛醌10，而携带dps基因在DDxxD结构域上突变的大肠杆菌JM109失去了产生泛醌10的能力，这表明根癌农杆菌dps基因在大肠杆菌中功能性表达，并且编码癸二烯基二磷酸合酶。

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