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Multigene expression of protein complexes by iterative modification of genomic Bacmid DNA.

Noad RJ, Stewart M, Boyce M, Celma CC, Willison KR, Roy P - BMC Mol. Biol. (2009)

Bottom Line: Use of bipartite selections can significantly improve selection of modified bacterial artificial chromosomes carrying baculovirus DNA.In addition to the commonly used p10 and polyhedrin loci, the ctx, egt, 39k, orf51, gp37, iap2 and odv-e56 loci in AcMNPV are all suitable for the high level expression of heterologous genes. 3.Two protein, four protein and eight protein complexes including virus-like particles and cellular chaperone complexes can be produced using the new approach.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK.

ABSTRACT

Background: Many cellular multi-protein complexes are naturally present in cells at low abundance. Baculovirus expression offers one approach to produce milligram quantities of correctly folded and processed eukaryotic protein complexes. However, current strategies suffer from the need to produce large transfer vectors, and the use of repeated promoter sequences in baculovirus, which itself produces proteins that promote homologous recombination. One possible solution to these problems is to construct baculovirus genomes that express each protein in a complex from a separate locus within the viral DNA. However current methods for selecting such recombinant genomes are too inefficient to routinely modify the virus in this way.

Results: This paper reports a method which combines the lambda red and bacteriophage P1 Cre-recombinase systems to efficiently generate baculoviruses in which protein complexes are expressed from multiple, single-locus insertions of foreign genes. This method is based on an 88 fold improvement in the selection of recombinant viruses generated by red recombination techniques through use of a bipartite selection cassette. Using this system, seven new genetic loci were identified in the AcMNPV genome suitable for the high level expression of recombinant proteins. These loci were used to allow the recovery two recombinant virus-like particles with potential biotechnological applications (influenza A virus HA/M1 particles and bluetongue virus VP2/VP3/VP5/VP7 particles) and the mammalian chaperone and cancer drug target CCT (16 subunits formed from 8 proteins).

Conclusion: 1. Use of bipartite selections can significantly improve selection of modified bacterial artificial chromosomes carrying baculovirus DNA. Furthermore this approach is sufficiently robust to allow routine modification of the virus genome. 2. In addition to the commonly used p10 and polyhedrin loci, the ctx, egt, 39k, orf51, gp37, iap2 and odv-e56 loci in AcMNPV are all suitable for the high level expression of heterologous genes. 3. Two protein, four protein and eight protein complexes including virus-like particles and cellular chaperone complexes can be produced using the new approach.

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Expression of virus like particles. A) Influenza A VLPs. Left and middle, stained SDS-PAGE and immunoblot, respectively, of cells expressing; Lane 1, Influenza HA (p10locus) and M1 (egt locus); Lane 2, Influenza HA (p10 locus) only; Lane 3, uninfected cells; Lane 4, Influenza M1 (egt locus). The immunoblot was probed with a polyclonal anti-influenza (H7N7) antibody. Right panel negative stain electron micrographs of influenza VLPs purified from the culture medium of infected cells. Bar = 50 nm. In the lower panel white arrows indicate the position of 5 nm gold particles labelling the HA spikes of the VLPs. B) Bluetongue virus VLPs. Left, stained SDS-PAGE of lysate from cells infected with AcMNPV expressing: Lane 1, BTV-1 VP5 (p10 locus); Lane 2, BTV-1VP2 (odv-e56 locus); Lane 3, BTV-1 VP3 (polyhedrin locus); Lane 4, BTV-1 VP7 (gp37 locus); Lane 5, Semipurified VLPs recovered from a virus coexpressing all 4 BTV proteins; M, marker lanes with proteins of defined mass. Right hand panels, electron micrographs of negatively stained BTV VLPs. Bar = 50 nm.
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Figure 4: Expression of virus like particles. A) Influenza A VLPs. Left and middle, stained SDS-PAGE and immunoblot, respectively, of cells expressing; Lane 1, Influenza HA (p10locus) and M1 (egt locus); Lane 2, Influenza HA (p10 locus) only; Lane 3, uninfected cells; Lane 4, Influenza M1 (egt locus). The immunoblot was probed with a polyclonal anti-influenza (H7N7) antibody. Right panel negative stain electron micrographs of influenza VLPs purified from the culture medium of infected cells. Bar = 50 nm. In the lower panel white arrows indicate the position of 5 nm gold particles labelling the HA spikes of the VLPs. B) Bluetongue virus VLPs. Left, stained SDS-PAGE of lysate from cells infected with AcMNPV expressing: Lane 1, BTV-1 VP5 (p10 locus); Lane 2, BTV-1VP2 (odv-e56 locus); Lane 3, BTV-1 VP3 (polyhedrin locus); Lane 4, BTV-1 VP7 (gp37 locus); Lane 5, Semipurified VLPs recovered from a virus coexpressing all 4 BTV proteins; M, marker lanes with proteins of defined mass. Right hand panels, electron micrographs of negatively stained BTV VLPs. Bar = 50 nm.

Mentions: To confirm that the system could successfully express and correctly assemble protein complexes, two well defined virus-like particle (VLP) systems were used as models. VLPs were produced for influenza A virus by co-expressing the M1 and HA proteins of the SC35M strain of influenza [31]. M1 was first inserted at the egt locus and then, following removal of the bipartite cassette, the HA gene was inserted into the p10 locus (Fig. 4A). Expression of both proteins was confirmed by SDS-PAGE and Immunoblot analysis (Fig. 4A, left and middle panels) using antibody specific for Influenza A (H7N7). Furthermore, it was possible to purify influenza VLPs from the culture medium of Sf9 cells infected with the recombinant baculovirus. The surface of these VLPs had the characteristic spike appearance of an enveloped virus (Fig. 4A, right panel) and it was possible to label these spike structures using immunogold labelling with anti-influenza (H7N7) antibody.


Multigene expression of protein complexes by iterative modification of genomic Bacmid DNA.

Noad RJ, Stewart M, Boyce M, Celma CC, Willison KR, Roy P - BMC Mol. Biol. (2009)

Expression of virus like particles. A) Influenza A VLPs. Left and middle, stained SDS-PAGE and immunoblot, respectively, of cells expressing; Lane 1, Influenza HA (p10locus) and M1 (egt locus); Lane 2, Influenza HA (p10 locus) only; Lane 3, uninfected cells; Lane 4, Influenza M1 (egt locus). The immunoblot was probed with a polyclonal anti-influenza (H7N7) antibody. Right panel negative stain electron micrographs of influenza VLPs purified from the culture medium of infected cells. Bar = 50 nm. In the lower panel white arrows indicate the position of 5 nm gold particles labelling the HA spikes of the VLPs. B) Bluetongue virus VLPs. Left, stained SDS-PAGE of lysate from cells infected with AcMNPV expressing: Lane 1, BTV-1 VP5 (p10 locus); Lane 2, BTV-1VP2 (odv-e56 locus); Lane 3, BTV-1 VP3 (polyhedrin locus); Lane 4, BTV-1 VP7 (gp37 locus); Lane 5, Semipurified VLPs recovered from a virus coexpressing all 4 BTV proteins; M, marker lanes with proteins of defined mass. Right hand panels, electron micrographs of negatively stained BTV VLPs. Bar = 50 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2749033&req=5

Figure 4: Expression of virus like particles. A) Influenza A VLPs. Left and middle, stained SDS-PAGE and immunoblot, respectively, of cells expressing; Lane 1, Influenza HA (p10locus) and M1 (egt locus); Lane 2, Influenza HA (p10 locus) only; Lane 3, uninfected cells; Lane 4, Influenza M1 (egt locus). The immunoblot was probed with a polyclonal anti-influenza (H7N7) antibody. Right panel negative stain electron micrographs of influenza VLPs purified from the culture medium of infected cells. Bar = 50 nm. In the lower panel white arrows indicate the position of 5 nm gold particles labelling the HA spikes of the VLPs. B) Bluetongue virus VLPs. Left, stained SDS-PAGE of lysate from cells infected with AcMNPV expressing: Lane 1, BTV-1 VP5 (p10 locus); Lane 2, BTV-1VP2 (odv-e56 locus); Lane 3, BTV-1 VP3 (polyhedrin locus); Lane 4, BTV-1 VP7 (gp37 locus); Lane 5, Semipurified VLPs recovered from a virus coexpressing all 4 BTV proteins; M, marker lanes with proteins of defined mass. Right hand panels, electron micrographs of negatively stained BTV VLPs. Bar = 50 nm.
Mentions: To confirm that the system could successfully express and correctly assemble protein complexes, two well defined virus-like particle (VLP) systems were used as models. VLPs were produced for influenza A virus by co-expressing the M1 and HA proteins of the SC35M strain of influenza [31]. M1 was first inserted at the egt locus and then, following removal of the bipartite cassette, the HA gene was inserted into the p10 locus (Fig. 4A). Expression of both proteins was confirmed by SDS-PAGE and Immunoblot analysis (Fig. 4A, left and middle panels) using antibody specific for Influenza A (H7N7). Furthermore, it was possible to purify influenza VLPs from the culture medium of Sf9 cells infected with the recombinant baculovirus. The surface of these VLPs had the characteristic spike appearance of an enveloped virus (Fig. 4A, right panel) and it was possible to label these spike structures using immunogold labelling with anti-influenza (H7N7) antibody.

Bottom Line: Use of bipartite selections can significantly improve selection of modified bacterial artificial chromosomes carrying baculovirus DNA.In addition to the commonly used p10 and polyhedrin loci, the ctx, egt, 39k, orf51, gp37, iap2 and odv-e56 loci in AcMNPV are all suitable for the high level expression of heterologous genes. 3.Two protein, four protein and eight protein complexes including virus-like particles and cellular chaperone complexes can be produced using the new approach.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK.

ABSTRACT

Background: Many cellular multi-protein complexes are naturally present in cells at low abundance. Baculovirus expression offers one approach to produce milligram quantities of correctly folded and processed eukaryotic protein complexes. However, current strategies suffer from the need to produce large transfer vectors, and the use of repeated promoter sequences in baculovirus, which itself produces proteins that promote homologous recombination. One possible solution to these problems is to construct baculovirus genomes that express each protein in a complex from a separate locus within the viral DNA. However current methods for selecting such recombinant genomes are too inefficient to routinely modify the virus in this way.

Results: This paper reports a method which combines the lambda red and bacteriophage P1 Cre-recombinase systems to efficiently generate baculoviruses in which protein complexes are expressed from multiple, single-locus insertions of foreign genes. This method is based on an 88 fold improvement in the selection of recombinant viruses generated by red recombination techniques through use of a bipartite selection cassette. Using this system, seven new genetic loci were identified in the AcMNPV genome suitable for the high level expression of recombinant proteins. These loci were used to allow the recovery two recombinant virus-like particles with potential biotechnological applications (influenza A virus HA/M1 particles and bluetongue virus VP2/VP3/VP5/VP7 particles) and the mammalian chaperone and cancer drug target CCT (16 subunits formed from 8 proteins).

Conclusion: 1. Use of bipartite selections can significantly improve selection of modified bacterial artificial chromosomes carrying baculovirus DNA. Furthermore this approach is sufficiently robust to allow routine modification of the virus genome. 2. In addition to the commonly used p10 and polyhedrin loci, the ctx, egt, 39k, orf51, gp37, iap2 and odv-e56 loci in AcMNPV are all suitable for the high level expression of heterologous genes. 3. Two protein, four protein and eight protein complexes including virus-like particles and cellular chaperone complexes can be produced using the new approach.

Show MeSH
Related in: MedlinePlus