Extracellular production of phospholipase A2 from Streptomyces violaceoruber by recombinant Escherichia coli.
Phospholipase A(2) (PLA(2)) from Streptomyces violaceoruber was successfully produced extracellularly in an active form by using a recombinant strain of Escherichia coli. The PLA(2) gene, which was artificially synthesized with optimized codons for E. coli and fused with pelB signal sequence, was expressed in E. coli using pET system. Most of the enzyme activity was detected in the culture supernatant with negligible activity in the cells. The recombinant enzyme was purified to homogeneity from the culture supernatant simply by ammonium sulfate precipitation and an anion exchange chromatography. The purified enzyme showed a specific activity comparable to that of the authentic enzyme. The recombinant enzyme had the same N-terminal amino acid sequence to that of the mature protein, indicating the correct removal of the signal peptide. An inactive PLA(2) with a mutation at the catalytic center was also secreted to the culture medium, suggesting that the observed secretion was not dependent on enzymatic activity. A simple screening method for the PLA(2)-producing colonies was established by detecting clear zone formation around the colonies on agar media containing lecithin. This is the first example of direct extracellular production of active PLA(2) by recombinant E. coli.
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan.
Daiki
D
Takemori
Kenta
K
Yoshino
Chisato
C
Eba
Hideo
H
Nakano
Yugo
Y
Iwasaki
D000645:Ammonium Sulfate / Q000378:metabolism; D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D001431:Bacteriological Techniques / Q000379:methods; D001483:Base Sequence; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange / Q000379:methods; D003470:Culture Media / Q000378:metabolism; D004789:Enzyme Activation; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D054467:Phospholipases A2 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D003470:Culture Media; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D054467:Phospholipases A2; D000645:Ammonium Sulfate