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.

Figure 1

Flowcharts for generation of recombinant viruses and protein production using Tn7-mediated transposition (a) or homologous recombination (b).

Figure 2

The HR-Bac toolbox components. (a) Schematic representation of the AcMNPV BAC10:KO₁₆₂₉, Δv-cath/chiA, mCherry bacmid highlighting (i) the modified v-cath/chiA locus where the mCherry-coding sequence was inserted to replace v-cath/chiA ORFs (left panel), and (ii) the inactivated PH locus in which a part of the essential orf1629 and the polyhedrin coding region were replaced by a bacterial replicon (lower panel). Homologous recombination with the transfer vector bearing the ORF of interest (green arrow) restores the function of orf1629 enabling virus replication and replaces the replicon sequence with the sequence(s) of interest. Sf9 cells infected with a recombinant virus generated using AcMNPV BAC10:KO₁₆₂₉ Δv-cath/chiA,mCherry viral DNA and a transfer vector containing the EGFP cDNA observed in a microscope (with a ×20 objective); mCherry and EGFP fluorescence for the same field are shown in the upper and lower panels, respectively. (b) Schematic representation of pAC8_GW and pAC8_MF transfer vectors. In pAC8_GW, cDNAs are inserted downstream of the polyhedrin (PH) promoter using a Gateway gene insertion cassette (GW). Proteins are expressed in fusion with an N-terminal affinity tag followed by a protease 3C cleavage site (red square) and with a C-terminal c-myc epitope. In pAC8_MF, cDNAs are expressed under the control of divergent PH and p10 promoters. The Ampicillin resistance gene (Amp R, black rectangles), the lef2,603 and orf1629 homology regions (AcMNPV, grey box) and the replication origin (black triangle) are indicated. A detailed map of the pAC8_MF expression cassette is shown in the lower panel. cDNAs are inserted using the BamHI/XbaI and NheI/XhoI restriction sites sequences. The PmeI and AvrII sequences as well as the BstZ17I/SpeI/NruI/ multiplication module from the MultiBac suite (M) are also depicted.

Figure 3

Production of binary complexes. (a) CDK9/cyclin T1. The cDNAs encoding Strep-CDK9 and cyclin T1 were cloned into the dual expression cassette of the pKL and pAC8_MF transfer vectors that were used to produce the complex with a virus obtained using the MultiBac toolkit (Tn7 mediated transposition, pK, lane 1) or virus generated with the HR-Bac toolbox (pAC8_MF, lane2). Affinity purified complexes from 10⁶ cells were analyzed using a Coomassie-stained 12.5% SDS gel. (b) XPG. Affinity purified Twin-strep tagged Komagataella phaffii (Pichia pastoris) XPG homologue produced using the pMF-mCherry/XPG transfer vector and analyzed using a Coomassie-stained 12.5% SDS gel. (c) RAR/RXR. Affinity purified Strep-tagged RAR/RXR heterodimer produced using the pMF-RAR/RXR transfer vector analyzed using a Coomassie-stained 12.5% SDS gel. (d) Schematic representation of the acceptor vector bearing the His-tagged PPAR coding sequence (light blue) (pAC8-His-PPAR) and the donor vector containing the RXR cDNA (dark blue) under the control of PH promoter. A fusion vector was obtained by Cre-mediated recombination using the lox P sites (green circles) of the acceptor and donor (left panel). Affinity purified His-tagged PPAR/RXR heterodimer produced from the fusion plasmid analyzed on a Coomassie-stained 12.5% SDS gel (right panel). Full-length gels are presented in Suppl. Fig. 6.

Figure 4

Production of ternary and quaternary complexes. (a) Organization of the CAK expression cassette (3900 bp). cDNAs encoding CDK7, cyclin H and MAT1 were assembled into pAC8_MF leading to pAC8_MF_CAK transfer plasmid where CDK7 expression is driven by the p10 promoter and where cyclin H and MAT1 are controlled by the PH promoter. Regions amplified (PCR1, 2500pb; PCR2, 1700pb; PCR3, 700pb) to control the integrity of the construct are also indicated (see c). (b) The pAC8_MF_CAK plasmid was co-transfected with four modified viral DNAs. Production of CAK from the different viruses was compared (lanes 1,2,3 and 4). (c) Baculoviral clones from a CAK expressing virus pool were isolated using plaque purification and amplified. Different PCRs from DNA of 24 viral clones were performed to control the integrity of the construct: PCR1 (2500pb, encompassing the orf1629 sequence and the CDK7 coding sequence), PCR2 (1700 bp, containing the lef2,603 sequence and MAT1 coding sequence) and PCR3 (targeting cyclin H coding sequence). PCR analysis showed that 17 out of the 20 resulting viruses (85%) have the expected complete structure. Here are only presented the 3 different PCR obtained for 2 clones (BV1 and BV2). The PCR products for all the other viral clones tested are shown as Suppl. Fig. 5b. Additionally, the ability of positive and negative baculoviral clones to produce the CAK subunits was evaluated by Western Blots (see Suppl. Fig. 5c). (d) Production of the quaternary CAK/XPD complex using one virus coding for four complex subunits. The fusion vector was generated through Lox mediated recombination between the pAC8_MF_CAK acceptor plasmid and the pSPL-DsRed-Flag-XPD donor vector. The complex was purified using FLAG (lane 1) followed by Strep (Lane 2) affinity chromatography. Full-length gels are shown in Suppl. Fig. 6.

Figure 5

The HR-bac toolbox. To assemble dual expression cassettes into transfer vectors, DNA elements comprising the plasmid backbone, cDNAs encoding the target genes and promoter modules (typically comprising the PH and p10 promoters) are combined in a single-step four-fragment homology-based assembly reaction. Expression cassettes can be excised by digestion with a pair of restriction endonucleases or amplified by PCR and inserted via compatible restriction sites or homology-based cloning into the multiplication module of a progenitor plasmid. An example in which an expression cassette containing genes C is cloned into a plasmid containing genes A and B is shown. Viruses are generated by co-transfection of insect cells with a transfer plasmid enclosing the expression cassette and a bacmid containing a defective version of the viral genome. As the bacmid is unable to initiate virus replication unless rescued by the transfer vector, the need for post-production screening is eliminated and recombinant viruses can be directly amplified, allowing to generate viruses from transfer vectors and express proteins in 2 weeks. Expression of a fluorescent marker proteins allows to monitor virus propagation. The full-length gel of the purified complex is shown in Suppl. Fig. 7a.