title: SweetBac: a new approach for the production of mammalianised glycoproteins in insect cells.
abstract: Recombinant production of therapeutically active proteins has become a central focus of contemporary life science research. These proteins are often produced in mammalian cells, in order to obtain products with post-translational modifications similar to their natural counterparts. However, in cases where a fast and flexible system for recombinant production of proteins is needed, the use of mammalian cells is limited. The baculoviral insect cell system has proven to be a powerful alternative for the expression of a wide range of recombinant proteins in short time frames. The major drawback of baculoviral systems lies in the inability to perform mammalian-like glycosylation required for the production of therapeutic glycoproteins. In this study we integrated sequences encoding Caenorhabditis elegans N-acetylglucosaminyltransferase II and bovine β1,4-galactosyltransferase I into the backbone of a baculovirus genome. The thereby generated SweetBac virus was subsequently used for the production of the human HIV anti-gp41 antibody 3D6 by integrating heavy and light chain open reading frames into the SweetBac genome. The parallel expression of target genes and glycosyltransferases reduced the yield of secreted antibody. However, the overall expression rate, especially in the recently established Tnao38 cell line, was comparable to that of transient expression in mammalian cells. In order to evaluate the ability of SweetBac to generate mammalian-like N-glycan structures on 3D6 antibody, we performed SDS-PAGE and tested for the presence of terminal galactose using Riccinus communis agglutinin I. The mammalianised variants of 3D6 showed highly specific binding to the lectin, indicating proper functionality. To confirm these results, PNGase A released N-glycans were analyzed by MALDI-TOF-MS and shown to contain structures with mainly one or two terminal galactose residues. Since the presence of specific N-glycans has an impact on antibodies ability to exert different effector functions, we tested the binding to human Fc gamma receptor I present on U937 cells.
authors.affiliation: Vienna Institute of BioTechnology - VIBT, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Dieter
authors.initials: D
authors.lastname: Palmberger
authors.forename: Iain B H
authors.initials: IB
authors.lastname: Wilson
authors.forename: Imre
authors.initials: I
authors.lastname: Berger
authors.forename: Reingard
authors.initials: R
authors.lastname: Grabherr
authors.forename: Dubravko
authors.initials: D
authors.lastname: Rendic
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D005700:Galactosyltransferases / Q000235:genetics; D005822:Genetic Vectors; D006023:Glycoproteins / Q000096:biosynthesis* / Q000378:metabolism; D006031:Glycosylation; D015483:HIV Antibodies / Q000096:biosynthesis*; D006801:Humans; D009036:Moths / Q000166:cytology*; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017452:Receptors, IgG / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D029742:Caenorhabditis elegans Proteins; D006023:Glycoproteins; D015483:HIV Antibodies; D017452:Receptors, IgG; D011994:Recombinant Proteins; D005700:Galactosyltransferases; D017351:N-Acetylglucosaminyltransferases; C063312:beta-1,3-galactosyl-0-glycosyl-glycoprotein beta-1,3-N-acetylglucosaminyltransferase