Abstract
A novel recombinant baculovirus expression vector was used to produce His-tagged human transferrin in a transformed insect cell line (Tn5beta4GalT) that constitutively expresses a mammalian beta-1,4-galactosyltransferase. This virus encoded the His-tagged human transferrin protein in conventional fashion under the control of the very late polyhedrin promoter. In addition, to enhance the synthesis of galactosylated biantennary N-glycans, this virus encoded human beta-1,2- N-acetylglucosaminyltransferase II under the control of an immediate-early (ie1) promoter. Detailed analyses by MALDI-TOF MS, exoglycosidase digestion, and two-dimensional HPLC revealed that the N-glycans on the purified recombinant human transferrin produced by this virus-host system included four different fully galactosylated, biantennary, complex-type glycans. Thus, this study describes a novel baculovirus-host system, which can be used to produce a recombinant glycoprotein with fully galactosylated, biantennary N-glycans.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Baculoviridae / genetics
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Carbohydrate Sequence
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Chromatography, High Pressure Liquid
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Enzyme-Linked Immunosorbent Assay
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Genetic Vectors
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Glycoside Hydrolases / metabolism
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Glycosylation
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Humans
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Molecular Sequence Data
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Monosaccharides / chemistry
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N-Acetylglucosaminyltransferases / genetics
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N-Acetylglucosaminyltransferases / metabolism*
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N-Acetyllactosamine Synthase / genetics
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N-Acetyllactosamine Synthase / metabolism*
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Nucleopolyhedroviruses / genetics
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Occlusion Body Matrix Proteins
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Oligosaccharides / chemistry*
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Promoter Regions, Genetic / genetics
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Spectrometry, Mass, Electrospray Ionization
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Spodoptera / enzymology*
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Spodoptera / genetics*
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Spodoptera / virology
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Transferrin / chemistry
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Transferrin / genetics
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Transferrin / metabolism*
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Viral Proteins / genetics
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Viral Proteins / metabolism
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Viral Structural Proteins
Substances
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Monosaccharides
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Occlusion Body Matrix Proteins
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Oligosaccharides
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Recombinant Proteins
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Transferrin
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Viral Proteins
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Viral Structural Proteins
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polyhedrin protein, Nucleopolyhedrovirus
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N-Acetylglucosaminyltransferases
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beta-1,3-galactosyl-0-glycosyl-glycoprotein beta-1,3-N-acetylglucosaminyltransferase
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N-Acetyllactosamine Synthase
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Glycoside Hydrolases