| Title |
Molecular Cloning of the Phospholipase D Gene from Streptomyces sp. YU100 and Its Expression in Escherichia coli |
| Author |
Ji-Seon Lee, Munkhtsetseg Bat-Ochir, Atanas V. Demirev, and Doo Hyun Nam* |
| Address |
Faculty of Pharmacy, Yeungnam University, Gyongsan 712-749, Republic of Korea |
| Bibliography |
Journal of Microbiology, 47(1),116-122, 2009,
|
| DOI |
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| Key Words |
phospholipase D, Streptomyces, gene cloning, heterologous expression |
| Abstract |
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. |
