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Titolo:
PREPARATION AND CHARACTERIZATION OF HUMAN RHEUMATOID ARTHRITIC SYNOVIAL-FLUID PHOSPHOLIPASE A(2) PRODUCED BY RECOMBINANT BACULOVIRUS-INFECTED INSECT CELLS
Autore:
KAWAUCHI Y; TAKASAKI J; MATSUURA Y; MASUHO Y;
Indirizzi:
YAMANOUCHI INST DRUG DISCOVERY RES,MOLEC MED RES LABS,21 MIYAKIGUOKA TSUKUBA IBARAKI 305 JAPAN NATL INST HLTH & NUTR,DEPT VET SCI,SHINJUKU KU TOKYO 162 JAPAN
Titolo Testata:
Journal of Biochemistry
fascicolo: 1, volume: 116, anno: 1994,
pagine: 81 - 87
SICI:
0021-924X(1994)116:1<81:PACOHR>2.0.ZU;2-G
Fonte:
ISI
Lingua:
ENG
Soggetto:
PANCREATIC PHOSPHOLIPASE-A2; EXPRESSION VECTORS; PURIFICATION; SECRETION; PROTEINS;
Keywords:
PHOSPHOLIPASE A(2); RECOMBINANT DNA; RHEUMATOID ARTHRITIS; SYNTHETIC GENE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
28
Recensione:
Indirizzi per estratti:
Citazione:
Y. Kawauchi et al., "PREPARATION AND CHARACTERIZATION OF HUMAN RHEUMATOID ARTHRITIC SYNOVIAL-FLUID PHOSPHOLIPASE A(2) PRODUCED BY RECOMBINANT BACULOVIRUS-INFECTED INSECT CELLS", Journal of Biochemistry, 116(1), 1994, pp. 81-87

Abstract

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 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 PLA, 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 PLA(2) activity into the culture medium. Theenzyme 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 therecombinant protein was recognized and cleaved at the signal sequencein 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.

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Documento generato il 04/04/20 alle ore 02:22:09