PREPARATION AND CHARACTERIZATION OF HUMAN RHEUMATOID ARTHRITIC SYNOVIAL-FLUID PHOSPHOLIPASE A(2) PRODUCED BY RECOMBINANT BACULOVIRUS-INFECTED INSECT CELLS

We prepared human rheumatoid arthritic synovial fluid phospholipase A( 2) (PLA(2)) [EC 3.1.1.4] from insect cells infected with a recombinant baculovirus. The PLA(2) DNA was designed, changing the original codon s to those used frequently in the polyhedrin gene. Sixteen oligo-deoxy nucleotides ranging from 40 to 70 nucleotides were chemically synthesi zed and then assembled to form the whole PLA, gene. The gene thus synt hesized was then placed under the control of the polyhedrin promoter o f Autographa californica nuclear polyhedrosis virus. The recombinant v irus was infected into Spodoptera frugiperda cells. The infected cells secreted protein having PLA(2) activity into the culture medium. The enzyme level in the medium reached about 3 mg/liter on day 4 after inf ection. The secreted protein was purified to a single band of 14,000 D a 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 e nzyme activity, substrate specificity, pH optimum, Ca2+ ion dependency , and kinetic values as those of the natural enzyme.