Glycosylation of a recombinant protein in the Tn5B1-4 insect cell line: influence of ammonia, time of harvest, temperature, and dissolved oxygen

Glycosylation is both cell line and protein dependent. Culture conditions can also influence the profile of glycoforms produced. To examine this possibility in the insect cell/baculovirus system, structures of N-linked oligosaccharides attached to SEAP (human secreted alkaline phosphatase), expressed under various culture conditions in BTI Tn5B1-4 cells, were characterized using FACE (fluorescence-assisted carbohydrate electrophoresis). Parameters varied were time of harvest, ammonia added during infection, dissolved oxygen, and temperature. It was found that glycosylation in the insect cell/baculovirus expression system is a robust, stable system that is less perturbed by variations in culture conditions than the level of protein expression. Addition of ammonia and low oxygen conditions affected SEAP expression, but not the oligosaccharide profile of SEAP. Time of SEAP harvest increased the amount of alpha-mannosidase resistant structures from 4.1% at 34 hours postinfection (h pi), to 5.0% at 100 h pi, and to 7.5% at 120 h pi. These structures were primarily sensitive to N-acetylhexosaminidase digest, although a small amount was insensitive to both mannosidase and N-acetyl-hexosaminidase digests. Lowering the temperature from 28 degrees C to 24 degrees C or even 20 degrees C, resulted in a twofold increase in oligosaccharides containing terminal alpha(1,3)-mannose residues. This condition did not affect the amount of mannosidase-resistant structures. However, this could result in more complete glycosylation of recombinant proteins in the BTI Tn5B1-4 cell line, because more structures with the potential for further processing would be produced.     

Maximizing production of estrogen receptor beta with the baculovirus expression system

Steroid hormone/nuclear receptor expression in cultured insect cell lines is routinely driven by a baculovirus vector. An advantage of the baculovirus production of these receptors is that large amounts of functional receptors are obtained for subsequent in vitro studies. Most laboratories produce nuclear receptors in Spodoptera frugiperda (Sf)9 cells. However, no one has determined whether this cell line is optimal for the production of any nuclear receptor. We compared the time course and level of estrogen receptor beta (ER beta) production from a baculovirus in two S. frugiperda cell lines, IPLB-SF21AE (Sf21) and Sf9, and two Trichloplusia ni cell lines, Tn368 and BTI-TN5b1-4 (High Five). Cells were harvested at various times (0.5-5 days) after infection. ER beta expression and activity was determined by specific [3H]estradiol (E2) binding, Western blot analysis, and estrogen response element (ERE) binding in vitro. The highest functional, bioactive ER beta expression both at the earliest time after infection and in the amount of ER beta produced/cell was with the Sf21 cell line. Baculovirus expressed ER beta-bound EREs with high affinity in a DNA sequence-dependent manner. We conclude that Sf21 cells are the best-suited cells for ER beta production.     

Use of mannosamine for inducing the addition of outer arm N-acetylglucosamine onto N-linked oligosaccharides of recombinant proteins in insect cells

Glycosylation is a cellular process accomplished by a series of sequential enzymatic processing steps through the endoplasmic reticulum and Golgi apparatus with vesicle transport between the membranous organelles. The capacity for complex glycosylation is considered to be conferred by cell genetics, while the roles of nongenetic factors in protein processing are often ignored. It was hypothesized that the glycosyltransferase reactions in the insect cell-baculovirus system were limited by the small supply of sugar donor cosubstrates. By adding mannosamine, the glycosylation of a human secreted alkaline phosphatase in Spodoptera frugiperda (Sf-21) cells was extended to include terminal N-acetylglucosamine structures which were not seen in control cultures, and in Trichoplusic ni (BTI-Tn5B1-4) cells the amount of terminal N-acetylglucosamine structures was increased.     

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 recombinant baculoviruses to produce authentic recombinant glycoproteins cloned from mammalian sources was investigated. A comparison was made of the N-linked glycans attached to secreted alkaline phosphatase (SEAP) produced in four species of insect larvae and their derived cell lines plus one additional insect cell line and larvae of one additional species. These data survey N-linked oligosaccharides produced in four families and six genera of the order Lepidoptera. Recombinant SEAP expressed by recombinant isolates of Autographa californica and Bombyx mori nucleopolyhedroviruses was purified from cell culture medium, larval hemolymph or larval homogenates by phosphate affinity chromatography. The N-linked oligosaccharides were released with PNGase-F, labeled with 8- aminonaphthalene-1-3-6-trisulfonic acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by fluorescence imaging. The oligosaccharide structures were confirmed with exoglycosidase digestions. Recombinant SEAP produced in cell lines of Lymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyx mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni , H.virescens , B.mori , and Danaus plexippus contained oligosaccharides that were structurally identical to the 10 oligosaccharides attached to SEAP produced in T.ni cell lines. The oligosaccharide structures were all mannose-terminated. Structures containing two or three mannose residues, with and without core fucosylation, constituted more than 75% of the oligosaccharides from the cell culture and larval samples.      

N-glycosylation of a baculovirus-expressed recombinant glycoprotein in three insect cell lines

Summary The capacity of two Trichoplusia ni (TN-368 and BTI-Tn-5bl-4) and a Spodoptera frugiperda (IPLB-SF-21A) cell lines to glycosylate recombinant, baculovirus-encoded, secreted, placental alkaline phosphatase was compared. The alkaline phosphatase from serum-containing, cell culture medium was purified by phosphate affinity column chromatography. The N-linked oligosaccharides were released from the purified protein with PNGase F and analyzed by fluorophore-assisted carbohydrate electrophoresis. The majority of oligosaccharide structures produced by the three cell lines contained two or three mannose residues, with and without core fucosylation, but there were structures containing up to seven mannose residues. The oligosaccharides that were qualitatively or quantitatively different between the cell lines were sequenced with glycosidase digestions. The S. frugiperda cells produced more fucosylated oligosaccharides than either of the T. ni cell lines. The smallest oligosaccharide produced by S. frugiperda cells was branched trimannose. In contrast, both T. ni cell lines produced predominantly dimannose and linear trimannose structures devoid of α 1–3-linked mannose.     

Lipophorin as a yolk precursor in Hyalophora cecropia: uptake kinetics and competition with vitellogenin

Vitellogenic follicles of Hyalophora cecropia were incubated in metabolically radiolabeled, high-density lipophorin isolated from pharate adult hemolymph by KBr density gradient centrifugation. The follicles transferred this probe from the incubation medium to the cortical yolk spheres in the oocyte by an energy-dependent and saturable mechanism. Vitellogenin and high-density lipophorin competed with each other for uptake, and are therefore concentrated by the follicle with a common mechanism. Microvitellin and lipophorin, in contrast, did not compete for uptake. The K(uptake) for the accumulation of high-density lipophorin was substantially higher than the value estimated earlier for vitellogenin (133 microM vs. 18 microM). This relationship helps explain why the shared concentrating mechanism does not deplete the lipid transport capacity of the hemolymph, and how a low vitellogenin: lipophorin molar ratio in the hemolymph yields a high ratio in the mature egg.