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Recombinant human secretory phospholipase A2: Purification and characterization of the enzyme for active site studies
- Source :
- Journal of Molecular Recognition. 5:145-153
- Publication Year :
- 1992
- Publisher :
- Wiley, 1992.
-
Abstract
- A secreted form of phospholipase A2 (PLA2) is thought to play an important role in inflammatory diseases. To characterize this enzyme the cDNA encoding a low molecular weight PLA2 was cloned from a human placental cDNA library. The cDNA encoding the human PLA2 was subcloned into an expression vector and subsequently transfected into Chinese hamster ovary (CHO) cells. A stable CHO cell clone, secreting ca 1 mg/L of recombinant PLA2 into the medium, was scaled up in culture to 180 L. The recombinant enzyme was purified from the cell supernatant to apparent homogeneity by a novel procedure combining adsorption to poly(vinylidene difluoride) membranes, ion exchange chromatography and size exclusion chromatography. The final recovery of PLA2 activity was 58%. A direct comparison between the purified recombinant human PLA2 and PLA2 purified from human synovial fluid, including molecular weight, antigenicity, ionic dependence, substrate specificity and sensitivity to known PLA2 inhibitors, indicated that the two enzymes exhibit identical biochemical properties. These results show that the recombinant PLA2 can be efficiently expressed and purified in sufficient quantities to characterize the enzyme active site, to aid in the rational development of PLA2 inhibitors as potential anti-inflammatory drugs, and to investigate further the role of PLA2 in inflammatory disease.
- Subjects :
- Antigenicity
Placenta
Immunoblotting
CHO Cells
Biology
Phospholipases A
Substrate Specificity
law.invention
Structural Biology
law
Cricetinae
Complementary DNA
Animals
Humans
Cloning, Molecular
Molecular Biology
chemistry.chemical_classification
Binding Sites
Expression vector
cDNA library
Chinese hamster ovary cell
Active site
Hydrogen-Ion Concentration
Chromatography, Ion Exchange
Molecular biology
Recombinant Proteins
Phospholipases A2
Enzyme
chemistry
Biochemistry
biology.protein
Recombinant DNA
Electrophoresis, Polyacrylamide Gel
lipids (amino acids, peptides, and proteins)
Subjects
Details
- ISSN :
- 09523499
- Volume :
- 5
- Database :
- OpenAIRE
- Journal :
- Journal of Molecular Recognition
- Accession number :
- edsair.doi.dedup.....0c824ed52594a07a2d35834b9ae713c5