MultiBac turns sweet

Abstract

The baculovirus/insect cell system has proven to be a powerful tool for the expression of eukaryotic proteins. Therapeutics, especially in the field of vaccinology, are often composed of several different protein subunits. Conventional baculoviral expression schemes largely lack efficient strategies for simultaneous multi-gene expression. The MultiBac technology which is based on an engineered genome of Autographa californica nuclear polyhedrosis virus in combination with specially designed transfer vectors is an elegant way for flexible generation of multi-subunit proteins in insect cells. Yet, the glycosylation pattern of insect cell-derived products is not favorable for many applications. Therefore, a modified version of MultiBac, SweetBac, was generated allowing for a flexible glycosylation of target proteins in insect cells. Beyond the SweetBac technology MultiBac can further be designed for bridging the gap between cell engineering and transient modulation of host genes for improved and product tailored expression of recombinant proteins.

Keywords: MultiBac; SweetBac; baculovirus; glycosylation; insect cells; virus-like particles.

Figure 1. The MultiBac vector system consists of an array of small synthetic DNA plasmids called acceptors and donors (A). Both classes contain a dual expression cassette (polh and p10) with eukaryotic polyadenylation signals (SV40 and HSVtk). Propagation of acceptors is driven by a regular origin of replication (ori^ColE1), whereas donors have a conditional origin (ori^R6Kγ) that requires special bacterial strains for replication. For selection in E.coli, acceptors contain a gentamycin resistance gene (Gn^R) and donors one antibiotic marker (Ab^R). For introduction in the MultiBac viral genome all vectors are equipped with a LoxP site and acceptors additionally with Tn7 transposition sites (Tn7L, Tn7R). Vectors are designed for an easy multiplexing of expression cassettes by the presence of specially designed restriction sites (B). Multigene constructs can futher be generated by fusing several donor vectors with one acceptor vector (C).

Figure 2. Multigene constructs based on acceptor vectors can be inserted in the MultiBac genome via the Tn7 transposition site. Within a second strategy expression cassettes can be inserted in the viral LoxP site in a Cre mediated reaction.

Figure 3. Schematic representation of the SweetBac platform. A donor vector containing open reading frames coding for Caenorhabditis elegans N-acetylglucosaminyltransferase II and the bovine β1,4-galactosyltransferase I was introduced in the LoxP site of a MultiBac genome resulting in SweetBac.