Molecular cloning of the phospholipase D gene from Streptomyces sp. YU100 and its expression in Escherichia coli.
The gene for phospholipase D (PLD) of Streptomyces sp. YU100 was cloned from lambda phage library and heterologously expressed in Escherichia coli. Using an amplified gene fragment based on the consensus sequences of streptomycetes PLDs, lambda 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.
Lee
Ji-Seon
JS
Faculty of Pharmacy, Yeungnam University, Gyongsan 712-749, Republic of Korea.
Bat-Ochir
Munkhtsetseg
M
Demirev
Atanas V
AV
Nam
Doo Hyun
DH
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial / Q000502:physiology*; D008969:Molecular Sequence Data; D010739:Phospholipase D / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics
D011994:Recombinant Proteins; D010739:Phospholipase D