Novel Baculovirus Expression Vectors That Provide Sialylation of Recombinant Glycoproteins in Lepidopteran Insect Cells

Jarvis, Donald L.; Howe, Dale; Aumiller, Jared J.

doi:10.1128/jvi.75.13.6223-6227.2001

Cited by 44 publications

(23 citation statements)

References 32 publications

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“…Recently, Jarvis and co-workers reported the occurrence of galactosylation and terminal sialylation on the baculoviral glycoprotein gp64 expressed by infection of Sf9 cells with a bovine galactosyltransferase/rat sialyltransferase dual recombinant baculovirus (35) or by infection of TN5 cells expressing both enzyme genes with a wild-type baculovirus (36). Collectively, the insufficient synthesis of sialylated N-glycans by insect cells may depend on, at least in part, the intense activity of insect-specific GlcNAcases and the significant but low activities of galactosyltransferase and sialyltransferase.…”

Section: Resultsmentioning

confidence: 99%

Sialylation of N-Glycans on the Recombinant Proteins Expressed by a Baculovirus-Insect Cell System under β-N-Acetylglucosaminidase Inhibition

Watanabe

Kokuho

Takahashi

et al. 2002

Journal of Biological Chemistry

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We investigated the ability of a baculovirus-insect cell system to produce sialylated glycoproteins. Despite the presence of enzymes for synthesizing complex-type Nglycans, the most frequent structure of insect N-glycan is the paucimannosidic type, Man 3 GlcNAc 2 (؎Fuc). The reason for the overwhelming assembly of paucimannosidic N-glycans is not yet well understood. We hypothesized that this predominance might be due to insectspecific, Golgi-associated ␤-N-acetylglucosaminidase (GlcNAcase)-mediated removal of N-acetylglucosamine residues from the precursor N-glycan, thereby preventing its galactosylation and terminal sialylation. As we expected, the suppression of intrinsic GlcNAcase activity with a specific inhibitor, 2-acetamido-1,2-dideoxynojirimycin, allowed the accumulation of sialylated glycoproteins in the supernatants of insect cell cultures after baculoviral infection. Our observation indicates that GlcNAcasedependent depletion of N-acetylglucosamine residues from intermediate N-glycans is critical for the assembly of paucimannosidic N-glycans in insect cells and, more importantly, that insect cells (under specific conditions) retain the ability to construct sialylated N-glycans like those in mammalian cells.

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Section: Resultsmentioning

confidence: 99%

Sialylation of N-Glycans on the Recombinant Proteins Expressed by a Baculovirus-Insect Cell System under β-N-Acetylglucosaminidase Inhibition

Watanabe

Kokuho

Takahashi

et al. 2002

Journal of Biological Chemistry

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“…The desired glycosylation patterns of glycoproteins can be achieved either by constructing a novel biosynthetic pathway through introducing genes encoding glycosyltransferases and glycosidases into the cells (61)(62)(63) or by blocking a specific biosynthesis activity through using antisense technology (64 y 65). Incorporation of two mammalian genes for a2, 6-sialyltransferase and βΐ, 4-galactosyltransferase into viral vectors has provided the lepidopteran insect cells with the new capability of sialylation of recombinant glycoproteins (66). Furthermore, overexpression of <x2,3-sialyltransferase and βΐ, 4-galactosyltransferase in CHO cells will result in maximizing sialic acid content of recombinant glycoproteins (67).…”

Section: Metabolic Engineeringmentioning

confidence: 98%

Fermentation Process Development of Carbohydrate-Based Therapeutics

Shu

2003

ACS Symposium Series

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This chapter focuses on the latest advances in the development of fermentation processes in carbohydrate-based therapeutics. Although fermentation has been the major route in producing bioactive compounds historically, the complexity of carbohydrates and the current remarkable accomplishments of glycobiology have forced a multidiscipline approach to the development of carbohydrate-based therapeutics. Current remarkable achievements of glycotechnology have been demonstrated in metabolic engineering, process optimization, analytical chemistry, monitoring techniques, enzymatic/ chemical synthesis, and separation techniques, etc. By intentionally incorporating these techniques into fermentation processes, numerous novel approaches have been generated to produce carbohydrate-based therapeutics with higher qualities in terms of authenticity and efficacy, and better yields. Besides, many novel bioactive compounds or drug candidates can be obtained by fermentation in a relatively economic approach.

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“…Subsequently, a recombinant baculovirus [131] and two transgenic insect cell lines (Sf 4GalT/ST6 [132] and Tn5 4GalT/ST6 [129]) were constructed carrying both 4GalT and a rat -2,6-sialyltransferase (St6gal1) in their genomes. This led to the production of sialylated N-glycans if the cells were grown in serumcontaining medium.…”

Section: N-glycan Engineering In Insect Cellsmentioning

confidence: 99%

N-glycosylation Engineering of Biopharmaceutical Expression Systems

Jacobs

Callewaert²

2009

CMM

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N-glycosylation, the enzymatic coupling of oligosaccharides to specific asparagine residues of nascent polypeptide chains, is one of the most widespread post-translational modifications. Following transfer of an N-glycan precursor in the ER, this structure is further modified by a number of glycosidases and glyco-syltransferases in the ER and the Golgi complex. The processing reactions occurring in the ER are highly conserved between lower and higher eukaryotes. In contrast, the reactions that take place in the Golgi complex are species- and cell type-specific. Due to its non-template driven nature, glycoproteins typically occur as a mixture of glycoforms. Since N-glycans influence circulation half-life, tissue distribution, and biological activity each glycoform has its own pharmacokinetic, pharmacodynamic and efficacy profile. Moreover, modification of glycoproteins with non-human oligosaccharides can result in undesired immunogenicity. Therefore, engineering of the N-glycosylation pathway of most currently used heterologous protein expression systems (bacteria, mammalian cells, insect cells, yeasts and plants) is actively pursued by several academic and industrial laboratories. These research efforts are in the first place directed at humanizing the N-glycosylation pathway and eliminating immunogenic glycotopes. Moreover, one wants to establish new structure-function relationships of different glycoforms, which helps to decreasing the complexity of the N-glycan repertoire towards one defined N-glycan structure. In this review, we discuss the most important recent milestones in the glycoengineering field.

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Novel Baculovirus Expression Vectors That Provide Sialylation of Recombinant Glycoproteins in Lepidopteran Insect Cells

Cited by 44 publications

References 32 publications

Sialylation of N-Glycans on the Recombinant Proteins Expressed by a Baculovirus-Insect Cell System under β-N-Acetylglucosaminidase Inhibition

Sialylation of N-Glycans on the Recombinant Proteins Expressed by a Baculovirus-Insect Cell System under β-N-Acetylglucosaminidase Inhibition

Fermentation Process Development of Carbohydrate-Based Therapeutics

N-glycosylation Engineering of Biopharmaceutical Expression Systems

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