N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells

The potential of insect cell cultures and larvae infected with recombinantbaculoviruses to produce authentic recombinant glycoproteins cloned frommammalian sources was investigated. A comparison was made of the N-linkedglycans attached to secreted alkaline phosphatase (SEAP) produced in fourspecies of insect larvae and their derived cell lines plus one additionalinsect cell line and larvae of one additional species. These data surveyN-linked oligosaccharides produced in four families and six genera of theorder Lepidoptera. Recombinant SEAP expressed by recombinant isolates ofAutographa californica and Bombyx mori nucleopolyhedroviruses was purifiedfrom cell culture medium, larval hemolymph or larval homogenates byphosphate affinity chromatography. The N-linked oligosaccharides werereleased with PNGase-F, labeled with 8- aminonaphthalene-1-3-6-trisulfonicacid, fractionated by polyacrylamide gel electrophoresis, and analyzed byfluorescence imaging. The oligosaccharide structures were confirmed withexoglycosidase digestions. Recombinant SEAP produced in cell lines ofLymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyxmori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni ,H.virescens , B.mori , and Danaus plexippus contained oligosaccharides thatwere structurally identical to the 10 oligosaccharides attached to SEAPproduced in T.ni cell lines. The oligosaccharide structures were allmannose-terminated. Structures containing two or three mannose residues,with and without core fucosylation, constituted more than 75% of theoligosaccharides from the cell culture and larval samples.