Influence of baculovirus-host cell interactions on complex N-linked glycosylation of a recombinant human protein.
The conditions required for mammalian-type complex N-linked glycosylation of human proteins produced in insect cells with the baculovirus expression vector system were investigated. Marked alterations to N-linked glycosylation of human placental secreted alkaline phosphatase (SEAP) were observed with different baculovirus species, insect cell lines, and cell culture media. When a recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) was used to produce SEAP in Trichoplusia ni (Tn-4h) cells cultured in serum-free medium, structural analyses indicated <1% hybrid and no complex oligosaccharides attached to SEAP, a typical result with the baculovirus expression vector system. However, when fetal bovine serum was added to the culture medium, 48 +/- 4% of the oligosaccharides were hybrid or complex (but asialylated) glycans. When a recombinant T. ni nucleopolyhedrovirus (TnSNPV) was similarly used to express SEAP in Tn-4h cells cultured in serum-containing medium, only 24 +/- 3% of the glycans contained terminal N-acetylglucosamine and/or galactose residues. In contrast, SEAP produced in Sf9 cells grown in serum-containing medium with AcMNPV contained <1% hybrid oligosaccharides and no complex oligosaccharides. The results illustrate that baculovirus type, host cell type, and the growth medium all have a strong influence on the glycosylation pathway in insect cells, resulting in significant alterations in structures and relative abundance of N-linked glycoforms. Although the addition of sialic acid residues to the SEAP glycans was not detected, possible approaches to obtain sialylated glycans are discussed.
Joshi
L
L
Boyce Thompson Institute at Cornell University, Tower Road, Ithaca, New York 14853, USA.
Davis
T R
TR
Mattu
T S
TS
Rudd
P M
PM
Dwek
R A
RA
Shuler
M L
ML
Wood
H A
HA
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000502:physiology*; D002851:Chromatography, High Pressure Liquid; D016895:Culture Media, Serum-Free; D006031:Glycosylation; D006801:Humans; D011134:Polysaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera / Q000821:virology*
D016895:Culture Media, Serum-Free; D011134:Polysaccharides; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase