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Preparation and characterization of human rheumatoid arthritic synovial fluid phospholipase A2 produced by recombinant baculovirus-infected insect cells.
- Source :
-
Journal of biochemistry [J Biochem] 1994 Jul; Vol. 116 (1), pp. 81-7. - Publication Year :
- 1994
-
Abstract
- We prepared human rheumatoid arthritic synovial fluid phospholipase A2 (PLA2) [EC 3.1.1.4] from insect cells infected with a recombinant baculovirus. The PLA2 DNA was designed, changing the original codons to those used frequently in the polyhedrin gene. Sixteen oligo-deoxynucleotides ranging from 40 to 70 nucleotides were chemically synthesized and then assembled to form the whole PLA2 gene. The gene thus synthesized was then placed under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus. The recombinant virus was infected into Spodoptera frugiperda cells. The infected cells secreted protein having PLA2 activity into the culture medium. The enzyme level in the medium reached about 3 mg/liter on day 4 after infection. The secreted protein was purified to a single band of 14,000 Da on SDS-PAGE, by means of cation exchange chromatography and reverse-phase HPLC. N-terminal amino acid sequence analysis revealed that the recombinant protein was recognized and cleaved at the signal sequence in the insect cell. The purified enzyme had almost the same specific enzyme activity, substrate specificity, pH optimum, Ca2+ ion dependency, and kinetic values as those of the natural enzyme.
- Subjects :
- Amino Acid Sequence
Animals
Base Sequence
Cell Line
DNA, Recombinant
Electrophoresis, Polyacrylamide Gel
Genetic Vectors
Humans
Molecular Sequence Data
Phospholipases A genetics
Phospholipases A2
Arthritis, Rheumatoid enzymology
Baculoviridae genetics
Phospholipases A biosynthesis
Spodoptera microbiology
Synovial Fluid enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 0021-924X
- Volume :
- 116
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Journal of biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 7798190
- Full Text :
- https://doi.org/10.1093/oxfordjournals.jbchem.a124507