Extracellular production of phospholipase A2 from Streptomyces violaceoruber by recombinant Escherichia coli
Autor: | Hideo Nakano, Chisato Eba, Yugo Iwasaki, Kenta Yoshino, Daiki Takemori |
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Rok vydání: | 2011 |
Předmět: |
Signal peptide
Genetic Vectors Molecular Sequence Data Biology Protein Sorting Signals medicine.disease_cause law.invention Bacterial Proteins law medicine Escherichia coli Chemical Precipitation Peptide sequence Ammonium sulfate precipitation Enzyme Assays chemistry.chemical_classification Phospholipase A Bacteriological Techniques Base Sequence Chromatography Ion Exchange Molecular biology Enzyme assay Recombinant Proteins Streptomyces Artificial Gene Fusion Culture Media Enzyme Activation Phospholipases A2 Enzyme Biochemistry chemistry Ammonium Sulfate Genes Bacterial Recombinant DNA biology.protein Biotechnology Plasmids |
Zdroj: | Protein expression and purification. 81(2) |
ISSN: | 1096-0279 |
Popis: | 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 . |
Databáze: | OpenAIRE |
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