Biochemical characterization of Drosophila melanogaster acetylcholinesterase expressed by recombinant baculoviruses.
Recombinant baculoviruses expressing full length and 3' truncated forms of c-DNA encoding the Drosophila melanogaster acetylcholinesterase (AChE) were constructed. Biochemical analyses showed that full length recombinant protein was enzymatically active and anchored to the cell membrane via a glycolipidic residue. DTT treatment dissociated the native form into monomers migrating as did the corresponding form of AChE extracted from drosophila heads. Finally, DFP labelling demonstrated that the specific proteolytic cleavage leading to the formation of 55 and 16 kDa subunits occurred in Sf9 cells. In contrast with the full-length enzyme, C-terminal-truncated forms were highly secreted, confirming the prominent role of the C-terminal hydrophobic peptide for the addition of the glycolipidic residue. Accumulation of inactive precursor was observed when recombinant proteins were overproduced using an improved baculovirus, suggesting a saturation of insect cell machineries.
Chaabihi
H
H
Unité de Biologie Cellulaire et Moléculaire, Laboratoire de Pathologie Comparée INRA-CNRS, Saint-Christol-Lez-Alès, France.
Fournier
D
D
Fedon
Y
Y
Bossy
J P
JP
Ravallec
M
M
Devauchelle
G
G
Cérutti
M
M
D000110:Acetylcholinesterase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D004331:Drosophila melanogaster / Q000201:enzymology*; D005822:Genetic Vectors; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013347:Subcellular Fractions / Q000201:enzymology / Q000648:ultrastructure; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000110:Acetylcholinesterase