Glycosylation and processing of high levels of active human glucocerebrosidase in invertebrate cells using a baculovirus expression vector.
A human cDNA containing the complete coding region for the lysosomal glycoprotein glucocerebrosidase (EC 3.2.1.45) was introduced into the genome of Autographa californica nuclear polyhedrosis virus downstream from the polyhedrin promoter. Infection of Spodoptera frugiperda cells (SF9) with recombinant virus produced high levels of glucocerebrosidase, 40% of which was in the culture medium. The amino-terminal amino acid sequence of the recombinantly produced enzyme was identical to that of mature, human placental glucocerebrosidase, demonstrating that the signal sequence of the human preenzyme was recognized and appropriately removed in the SF9 invertebrate cells. The glucocerebrosidase in both the culture supernatant and SF9 cell pellet was glycosylated and contained, in part, high mannose oligosaccharide. These results demonstrate that insect cells can be used to produce abundant quantities of active mature human glucocerebrosidase that contains high mannose oligosaccharide as a consequence of post-translational processing.
Martin
B M
BM
Molecular Neurogenetics Section, National Institute of Mental Helath, Bethesda, MD 20892.
Tsuji
S
S
LaMarca
M E
ME
Maysak
K
K
Eliason
W
W
Ginns
E I
EI
D000818:Animals; D004274:DNA, Recombinant; D005822:Genetic Vectors; D005959:Glucosidases / Q000235:genetics*; D005962:Glucosylceramidase / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007120:Immunochemistry; D007304:Insect Viruses / Q000235:genetics*; D007313:Insecta; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D005959:Glucosidases; D005962:Glucosylceramidase