Preparation and characterization of human rheumatoid arthritic synovial fluid phospholipase A2 produced by recombinant baculovirus-infected insect cells.
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.
Kawauchi
Y
Y
Molecular Medicine Research Laboratories, Yamanouchi Institute for Drug Discovery Research, Ibaraki.
Takasaki
J
J
Matsuura
Y
Y
Masuho
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001172:Arthritis, Rheumatoid / Q000201:enzymology*; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000096:biosynthesis* / Q000235:genetics; D054467:Phospholipases A2; D018411:Spodoptera / Q000382:microbiology*; D013582:Synovial Fluid / Q000201:enzymology*
D004274:DNA, Recombinant; D010741:Phospholipases A; D054467:Phospholipases A2