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Cloning and Functional Expression of the dps Gene Encoding Decaprenyl Diphosphate Synthase from Agrobacterium tumefaciens
Author(s) -
Lee JungKul,
Her Gun,
Kim SangYong,
Seo JinHo
Publication year - 2008
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp034213e
Subject(s) - agrobacterium tumefaciens , biology , biochemistry , open reading frame , gene , atp synthase , microbiology and biotechnology , peptide sequence , escherichia coli , nucleic acid sequence , molecular cloning , amino acid , transgene
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.