- Molecular Cloning of the Phospholipase D Gene from Streptomyces sp. YU100 and Its Expression in Escherichia coli
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Ji-Seon Lee , Munkhtsetseg Bat-Ochir , Atanas V. Demirev , Doo Hyun Nam
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J. Microbiol. 2009;47(1):116-122. Published online February 20, 2009
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DOI: https://doi.org/10.1007/s12275-008-0161-8
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The gene for phospholipase D (PLD) of Streptomyces sp. YU100 was cloned from λ phage library and heterologously expressed in Escherichia coli. Using an amplified gene fragment based on the consensus sequences of streptomycetes PLDs, λ phage library of Streptomyces sp. YU100 chromosomal DNA was screened. The sequencing result of BamHI-digested 3.8 kb fragment in a positive phage clone revealed the presence of an open reading frame of a full sequence of PLD gene encoding a 540-amino acid protein including 33-amino acid signal peptide. The deduced amino acid sequence showed a high homology with other Streptomyces PLDs, having the highly conserved ‘HKD’ motifs. The PLD gene excluding signal peptide sequence was amplified and subcloned into a pET-32b(+) expression vector in E. coli BL21(DE3). The recombinant PLD was purified by nickel affinity chromatography and compared the enzyme activity with wild-type PLD. The results imply that the recombinant PLD produced by E. coli had the nearly same enzyme activity as PLD from Streptomyces sp. YU100.
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Citations
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