Complex-type biantennary N-glycans of recombinant human transferrin from Trichoplusia ni insect cells expressing mammalian [beta]-1,4-galactosyltransferase and [beta]-1,2-N-acetylglucosaminyltransferase II.
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.
Tomiya
Noboru
N
Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA. ntomiyal@jhu.edu
Howe
Dale
D
Aumiller
Jared J
JJ
Pathak
Manuj
M
Park
Jung
J
Palter
Karen B
KB
Jarvis
Donald L
DL
Betenbaugh
Michael J
MJ
Lee
Yuan C
YC
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000737:chemistry; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D009237:N-Acetyllactosamine Synthase / Q000235:genetics / Q000378:metabolism*; D017924:Nucleopolyhedroviruses / Q000235:genetics; D000077183:Occlusion Body Matrix Proteins; D009844:Oligosaccharides / Q000737:chemistry*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera / Q000201:enzymology* / Q000235:genetics* / Q000821:virology; D014168:Transferrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D015678:Viral Structural Proteins
D009005:Monosaccharides; D000077183:Occlusion Body Matrix Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014168:Transferrin; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus; D017351:N-Acetylglucosaminyltransferases; C063312:beta-1,3-galactosyl-0-glycosyl-glycoprotein beta-1,3-N-acetylglucosaminyltransferase; D009237:N-Acetyllactosamine Synthase; D006026:Glycoside Hydrolases