|
KMID : 0880220090470010116
|
|
Journal of Microbiology
2009 Volume.47 No. 1 p.116 ~ p.122
|
|
|
Molecular Cloning of the Phospholipase D Gene from Streptomyces sp. YU100 and Its Expression in Escherichia coli
|
|
Lee Ji-Seon
Munkhtsetseg Bat-Ochir
Atanas V. Demirev
Nam Doo-Hyun
|
|
|
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.
|
|
|
KEYWORD
|
|
|
phospholipase D, Streptomyces, gene cloning, heterologous expression
|
|
|
FullTexts / Linksout information
|
|
|
|
|
|
Listed journal information
|
|
  
|