title: A novel baculovirus vector for the production of nonfucosylated recombinant glycoproteins in insect cells.
abstract: Glycosylation is an important attribute of baculovirus-insect cell expression systems, but some insect cell lines produce core α1,3-fucosylated N-glycans, which are highly immunogenic and render recombinant glycoproteins unsuitable for human use. To address this problem, we exploited a bacterial enzyme, guanosine-5'-diphospho (GDP)-4-dehydro-6-deoxy-d-mannose reductase (Rmd), which consumes the GDP-l-fucose precursor. We expected this enzyme to block glycoprotein fucosylation by blocking the production of GDP-l-fucose, the donor substrate required for this process. Initially, we engineered two different insect cell lines to constitutively express Rmd and isolated subclones with fucosylation-negative phenotypes. However, we found the fucosylation-negative phenotypes induced by Rmd expression were unstable, indicating that this host cell engineering approach is ineffective in insect systems. Thus, we constructed a baculovirus vector designed to express Rmd immediately after infection and facilitate the insertion of genes encoding any glycoprotein of interest for expression later after infection. We used this vector to produce a daughter encoding rituximab and found, in contrast to an Rmd-negative control, that insect cells infected with this virus produced a nonfucosylated form of this therapeutic antibody. These results indicate that our Rmd(+) baculoviral vector can be used to solve the immunogenic core α1,3-fucosylation problem associated with the baculovirus-insect cell system. In conjunction with existing glycoengineered insect cell lines, this vector extends the utility of the baculovirus-insect cell system to include therapeutic glycoprotein production. This new vector also extends the utility of the baculovirus-insect cell system to include the production of recombinant antibodies with enhanced effector functions, due to its ability to block core α1,6-fucosylation.
authors.affiliation: Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Mabashi-Asazuma
authors.forename: Chu-Wei
authors.initials: CW
authors.lastname: Kuo
authors.forename: Kay-Hooi
authors.initials: KH
authors.lastname: Khoo
authors.forename: Donald L
authors.initials: DL
authors.lastname: Jarvis
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D001709:Biotechnology / Q000379:methods; D005643:Fucose / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D001426:Bacterial Proteins; D006023:Glycoproteins; D011994:Recombinant Proteins; D005643:Fucose; D007658:Ketone Oxidoreductases; C449658:GDP-4-keto-6-deoxy-D-mannose reductase
keywords: baculovirus; fucosylation; glycoengineering; glycoprotein production; insect cells