Secretion of a functional soluble form of neutral endopeptidase-24.11 from a baculovirus-infected insect cell line.
Neutral endopeptidase (NEP; EC 3.4.24.11) is an integral membrane protein found at the plasma membrane of many cell types. A secreted form of NEP (sec-NEP) was recently obtained by transfection of COS-1 cells with a recombinant expression vector consisting of the cDNA encoding the signal peptide of pro-opiomelanocortin fused in-frame to the cDNA sequence of the complete ectodomain of rabbit NEP [Lemay, Waksman, Roques, Crine & Boileau (1989) J. Biol. Chem. 264, 15620-15623]. In order to produce large quantities of this enzyme for structural studies we have expressed this recombinant soluble form of NEP at high yields using a baculovirus/insect-cell system. A recombinant Autographa californica nuclear polyhedrosis-virus genome containing the sec-NEP sequence was used to infect host Spodoptera frugiperda Sf9 cells. Infected cells secreted an N-glycosylated soluble form of neutral endopeptidase which was enzymically active. The yield was about 80 nmol of enzyme/litre of culture. The soluble form of the recombinant enzyme purified by immunoaffinity showed the same catalytic properties as the wild-type enzyme extracted from the kidney brush-border membranes. Treatment of the recombinant enzyme with endo-beta-N-acetylglucosaminidase H showed, however, that invertebrate cells did not glycosylate the enzyme to the same extent as did mammalian cells. Our findings demonstrate that insect cells can be used as hosts for the production of the soluble form of neutral endopeptidase. We also conclude that neither a full complement of carbohydrate side chains nor the membrane anchor appear to be essential for the production and targeting to the cell surface of a fully functional enzyme in this expression system.
Fossiez
F
F
Département de Biochimie, Faculté de Médecine, Université de Montréal, Québec, Canada.
Lemay
G
G
Labonté
N
N
Parmentier-Lesage
F
F
Boileau
G
G
Crine
P
P
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors / Q000502:physiology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009036:Moths / Q000378:metabolism* / Q000382:microbiology; D015260:Neprilysin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011333:Pro-Opiomelanocortin / Q000378:metabolism; D021382:Protein Sorting Signals / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D011333:Pro-Opiomelanocortin; D004247:DNA; D015260:Neprilysin