N-glycan patterns of human transferrin produced in Trichoplusia ni insect cells: effects of mammalian galactosyltransferase.
(2000)
Journal - Glycobiology (ENGLAND )
Abstract :
The N-glycans of human serum transferrin produced in Trichopulsia ni cells were analyzed to examine N-linked oligosaccharide processing in insect cells. Metabolic radiolabeling of the intra- and extracellular protein fractions revealed the presence of multiple transferrin glycoforms with molecular weights lower than that observed for native human transferrin. Consequently, the N-glycan structures of transferrin in the culture medium were determined using three-dimensional high performance liquid chromatography. The attached oligosaccharides included high mannose, paucimannosidic, and hybrid structures with over 50% of these structures containing one fucose, alpha(1,6)-, or two fucoses, alpha(1,6)- and alpha(1,3)-, linked to the Asn-linked N-acetylglucosamine. Neither sialic acid nor galactose was detected on any of the N-glycans. However, when transferrin was coexpressed with beta(1,4)-galactosyltransferase three additional galactose-containing hybrid oligosaccharides were obtained. The galactose attachments were exclusive to the alpha(1, 3)-mannose branch and the structures varied by the presence of zero, one, or two attached fucose residues. Furthermore, the presence of the galactosyltransferase appeared to reduce the number of paucimannosidic structures, which suggests that galactose attachment inhibits the ability of hexosaminidase activity to remove the terminal N-acetylglucosamine. The ability to promote galactosylation and reduce paucimannosidic N-glycans suggests that the oligosaccharide processing pathway in insect cells may be manipulated to mimic more closely that of mammalian cells.
| ISSN : | 0959-6658 |
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| Mesh Heading : | Animals Base Sequence Carbohydrate Conformation Carbohydrate Sequence Chromatography, High Pressure Liquid DNA Primers Galactosyltransferases Humans Molecular Sequence Data Moths Polysaccharides Recombinant Proteins Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Transferrin Tunicamycin chemistry genetics metabolism chemistry genetics pharmacology |
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| Mesh Heading Relevant : | metabolism cytology metabolism metabolism |
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A bacterial signal peptidase enhances processing of a recombinant single chain antibody fragment in insect cells.
(1999)
Journal - Biochemical and biophysical research communications (UNITED STATES )
Abstract :
The production of an antibody single chain fragment (scFv) in insect cells was accompanied by the formation of an insoluble intracellular precursor even with the inclusion of the bee melittin signal peptide. The presence of the precursor polypeptide suggests a limitation in the processing of the signal peptide so a baculovirus containing a signal peptidase from Bacillus subtilis (SipS) was constructed for expression studies. When the wild type SipS was coexpressed with scFv, preprocessed scFv fragments were no longer detected in insect cell lysates. Conversely, coexpression of scFv alone or with an inactive mutant SipS resulted in at least 30% of the intracellular polypeptide in an unprocessed form at 3 days post infection. Production of scFv in the medium was also enhanced in the presence of SipS; however, low secretion levels indicate the presence of a post-processing bottleneck.Copyright 1999 Academic Press.
| ISSN : | 0006-291X |
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| Mesh Heading : | Animals Bacillus subtilis Bacterial Proteins Baculoviridae Cell Line Cell Survival Genetic Vectors Immunoglobulin Variable Region Mice Peptide Fragments Protein Processing, Post-Translational Recombinant Proteins Serine Endopeptidases Spodoptera enzymology biosynthesis genetics genetics metabolism metabolism genetics metabolism biosynthesis biosynthesis genetics cytology enzymology |
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| Mesh Heading Relevant : | Membrane Proteins physiology genetics genetics metabolism physiology genetics |
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Overexpression of a cytosolic chaperone to improve solubility and secretion of a recombinant IgG protein in insect cells.
(1999)
Journal - Biotechnology and bioengineering (UNITED STATES )
Abstract :
The secretion of heterologous IgG proteins in the baculovirus-insect cell expression system is accompanied by substantial insoluble immunoglobulin in the infected cells. The accumulation of these insoluble forms suggests a limitation in the processing and secretory pathway of the infected cells. As a result, cytosolic hsp70 chaperones, which are known to associate and prevent aggregation of polypeptides in vitro, have been coexpressed in the infected cells. The hsp70 protein coprecipitated with the immunoglobulin to indicate the formation of a specific hsp70-immunoglobulin complex in vivo. Immunoblot and pulse chase studies indicated that coexpression of hsp70 increased intracellular immunoglobulin solubility. Metabolic labeling experiments revealed that hsp70 increased secreted immunoglobulin levels after several days infection as compared to infection with control baculoviruses. Pulse chase studies indicated that hsp70 increases the solubility of immunoglobulin precursors that are then processed and assembled into the complete antibody oligomer. A comparison of the action of cytosolic hsp70 chaperone to the endoplasmic reticulum chaperone BiP suggests sequential action in which hsp70 increases the solubility of preprocessed immunoglobulin, while BiP enhances the solubility of processed immunoglobulin chains.Copyright 1998 John Wiley & Sons, Inc.
| ISSN : | 0006-3592 |
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| Mesh Heading : | Animals Blotting, Western Carrier Proteins Cells, Cultured Cytosol Densitometry Endoplasmic Reticulum HSP70 Heat-Shock Proteins Immunoglobulin G Molecular Chaperones Moths Protein Folding Recombinant Proteins Solubility biosynthesis genetics metabolism metabolism genetics genetics biosynthesis genetics |
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| Mesh Heading Relevant : | Heat-Shock Proteins biosynthesis biosynthesis secretion |
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