HR-Bac, a toolbox based on homologous recombination for expression, screening and production of multiprotein complexes using the baculovirus expression system

Abstract

The Baculovirus/insect cell expression system is a powerful technology for reconstitution of eukaryotic macromolecular assemblies. Most multigene expression platforms rely on Tn7-mediated transposition for transferring the expression cassette into the baculoviral genome. This allows a rigorous characterization of recombinant bacmids but involves multiple steps, a limitation when many constructs are to be tested. For parallel expression screening and potential high throughput applications, we have established an open source multigene-expression toolbox exploiting homologous recombination, thus reducing the recombinant baculovirus generation to a single-step procedure and shortening the time from cloning to protein production to 2 weeks. The HR-bac toolbox is composed of a set of engineered bacmids expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors. They contain single or dual expression cassettes bearing different affinity tags and their design facilitates the mix and match utilization of expression units from Multibac constructs. The overall cost of virus generation with HR-bac toolbox is relatively low as the preparation of linearized baculoviral DNA only requires standard reagents. Various multiprotein assemblies (nuclear hormone receptor heterodimers, the P-TEFb or the ternary CAK kinase complex associated with the XPD TFIIH subunit) are used as model systems to validate the toolbox presented.

A lipid transfer protein ensures nematode cuticular impermeability

“…The pAC8_MF_GMAP-1 transfer vector was co-transfected in Sf9 cells with linearized AcMNPV viral DNA (Bac10:KO 1629, Δv-cath/chiA-LoxP:dsRed) to generate the recombinant baculovirus ( Kolesnikova et al., 2022 ) and the corresponding virus was amplified. For protein production, Hi Five cells cultivated in Express-five serum free medium were infected at a density of 1.0 × 10 6 cells/mL and MOI of 2 (Detailed protocols for baculovirus expression can be found in ( Osz-Papai et al., 2015 ).…”

“…The following references appear in the supplemental information: Schindelin et al. (2012) ; Kolesnikova et al. (2022) .…”

Genetic engineering of baculovirus-insect cell system to improve protein production

“…HR-bac, a toolbox based on MultiBac, composed of a set of engineered bacmids capable of expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors. This design facilitates expression screening and potential high-throughput applications ( Kolesnikova et al, 2022 ). Another system, biGBac, is a simple, efficient and rapid baculovirus vector system that could be used to construct multigene expression.…”

A Lipid Transfer Protein Ensures Nematode Cuticular Impermeability

“…Supporting citations: the following references appear in the Supplemental information (Schindelin et al, 2012), (Kolesnikova et al, 2022).…”