Katharina Paschinger for her glycomic expertise and reading the manuscript as well as Dr. an impact on antibodies ability to exert different effector functions, we tested the binding to human Fc gamma receptor I present on U937 cells. Introduction The use of insect cells for the production of therapeutically active proteins has gained increasing importance over the last decade, highlighted by the market entry of the human papilloma virus vaccine Cervarix? [1]. Nowadays, a set of different insect cell lines, suitable for the baculovirus-driven production of recombinant proteins, is available. 20-HETE The most popular ones are BTI-TN5B1-4 High Five (Hi5) cells [4]. More recently, a new cell line, BTI-have been demonstrated to produce significantly higher amounts in many cases [15], [16]. Therefore, we decided to revive the idea of using the baculovirus itself for glycoengineering purposes. The MultiBac platform, which is an advanced version of the Bac-to-Bac system, provides the possibility of flexible multigene expression using a single baculovirus vector [17]C[20]. In this study we evaluated the use of MultiBac for the generation of an efficient platform for the production of properly glycosylated proteins in lepitopteran insect cells other than the commonly used BTI-TN5B1-4 High Five (Hi5) cells (ATCC CRL-10859) [4] and BTI-nucleopolyhedroviruses were isolated and plaque purified by standard procedures. Viral titres were determined by plaque assay using 10-fold dilution series (n?=?3). Construction of SweetBac The agglutinin I (RCA I) (Vector Labs, B-1085) 20-HETE in TPBS (PBS+0.05% Tween 20) and streptavidin-conjugated alkaline phosphatase (Vector-Labs, SA-5100). The blot was developed using NBT/BCIP Sigma fast tablets (Sigma, B5655). N-glycan analysis N-glycan analyses were essentially performed as described in Rendic et al. [24]. Briefly, after the heavy and light chains of purified IgG1 were separated on an SDS-PAGE, bands corresponding to heavy chains were excised. After washing the bands, they were treated with dithiothreitol and iodoacetamide solutions to change cysteine residues. The GNG4 gel bands were washed again and then subjected to trypsin digestion at 37C over night. The peptides extracted with AcNH2OTFA solution (acetonitrile/water/trifluoroacetic acid; 6663331) were then dried, resuspended in 20 l of 50 mM ammonium acetate (pH 5) buffer and subjected to PNGase A treatment at 37C over night. The released N-glycans were separated from 20-HETE peptides by using columns packed with 10 L LiChroprep RP 18 (25C40 M) reversed phase resin (Merck) on top of 40 L Dowex 50WX8-400 ion exchange resin (Sigma, 217514). After equilibrating the column with 2% acetic acid, the sample was applied, the column was washed with 2% acetic acid and the flow-through made up of N-glycans was collected. For further purification of the released N-glycans, columns were packed with Supelclean? ENVI-Carb? PGC material (Sigma) on top of LiChroprep RP 18 (25C40 M) reversed phase resin (Merck) and equilibrated with 2% acetic acid. The samples were loaded around the column and after washing with H2O, the N-glycans were eluted with 40% acetonitrile. The purified N-glycans were dried in a SpeedVac, finally resuspended in 5 L deionised water and used for MALDI-TOF-MS analysis (on a Bruker Ultraflex MALDI-TOF/TOF in positive reflectron mode) using 6-aza-2-thiothymine (ATT) as matrix. Flow cytometric analysis GnTII and a bovine GalT 20-HETE controlled by p10 and polyhedrin (ph) promoter respectively. This module was integrated into the loxP site of a MultiBac genome. The resulting SweetBac was then used as a basis for the production of recombinant glycoproteins with mammalianised complex type N-glycans. In order to evaluate the functionality of SweetBac, we additionally introduced heavy (HC) and light chain (LC) open reading frames of the human HIV anti-gp41 antibody 3D6 [22] in the Tn7 site of a SweetBac genome, resulting in SweetBac-3D6. As a negative control, open reading frames coding the heavy and the light chains of the same antibody were integrated in a wild-type MultiBac genome as well (Physique 1). Open in a separate window Physique 1.