Human group II phospholipase A2 expressed in Trichoplusia ni larvae--isolation and kinetic properties of the enzyme.
Human secreted synovial fluid/platelet-type group II phospholipase A2 (sPLA2) was expressed in Trichoplusia ni (cabbage looper) larvae and cultured Sf9 insect cells by infection with a recombinant baculovirus. Active sPLA2, with correct N-terminal proteolytic processing, was not secreted by Sf9 cells in culture. The enzyme was isolated from their homogenate without any need for refolding or renaturation of the protein. The enzyme was extracted from the 5000g pellet with 1 M KBr and isolated by chromatography on a cation exchange column followed by reverse-phase chromatography on a Butyl Aquapore column. The yield of active enzyme (25 micrograms/g insect) was comparable to yields obtained in CHO cells or Escherichia coli by other investigators. The recombinant enzyme had the correct N-terminal sequence, expected molecular weight, and reacted with antisera raised against peptides inferred from the cDNA sequence of the natural enzyme. Monoclonal antibodies were raised against the recombinant sPLA2 and they permitted the isolation of the natural enzyme from human serum by immunoaffinity. The recombinant sPLA2 showed a preference for substrate vesicles with a net negative charge. The baculovirus expression system provided active sPLA2 that can be produced economically in insects, purified simply, had well-defined kinetic properties, and should be useful in studies of inflammatory disorders.
Tremblay
N M
NM
Merck Frosst Centre for Therapeutic Research, Merck Frosst Canada, Inc., Dorval, Québec.
Kennedy
B P
BP
Street
I P
IP
Kaupp
W J
WJ
Laliberté
F
F
Weech
P K
PK
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification; D002460:Cell Line; D002846:Chromatography, Affinity; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D007814:Larva; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009036:Moths / Q000145:classification / Q000254:growth & development / Q000378:metabolism*; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D010741:Phospholipases A / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D054467:Phospholipases A2; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D010741:Phospholipases A; D054467:Phospholipases A2