Lipase from Pseudomonas aeruginosa. Production in Escherichia coli and activation in vitro with a protein from the downstream gene.
The lipase gene from Pseudomonas aeruginosa TE3285 is followed by another gene, lipB. The lipase gene was expressed in Escherichia coli BL21(DE3)pLysS using the T7 RNA polymerase expression system. The mature lipase was accumulated as inclusion bodies at 42% of the total cell proteins. The inclusion bodies were solubilized with 8 M urea, but lipase activity was not detected in the solubilized preparation containing 85% lipase protein even after removing urea by dialysis. The lipB gene, positioned downstream of the lipase gene and thought to be necessary for the expression of the lipase gene, was expressed in Escherichia coli JM109 as a fusion with the glutathione transferase gene from Schistosoma japonicum. The fusion protein was partially purified on glutathione-agarose beads to 36% purity. Incubated with the fusion protein at a molar ratio of 1:1 at 4 degrees C for 24 h, the solubilized lipase showed lipase activity of about a tenth that of the purified lipase prepared from Pseudomonas aeruginosa TE3285. Magnesium ions and ATP were not essential but increased the activation. When the fusion protein was treated with thrombin to release the glutathione transferase part, it retained its activity. The lipase activation with lipB protein probably proceeds to form a 1:1 complex with the inactive, solubilized lipase protein but by a different mode from known chaperones.
Oshima-Hirayama
N
N
Institute for Chemical Research, Kyoto University, Japan.
Yoshikawa
K
K
Nishioka
T
T
Oda
J
J
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D008049:Lipase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012995:Solubility; D013917:Thrombin / Q000494:pharmacology
D001426:Bacterial Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D008049:Lipase; D013917:Thrombin; D008274:Magnesium