Abstract
BackgroundMany 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.ResultsThis 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).Conclusion1. 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.
Highlights
Many cellular multi-protein complexes are naturally present in cells at low abundance
Four protein and eight protein complexes including virus-like particles and cellular chaperone complexes can be produced using the new approach
The Autographa califonica multiple nucleopolyhedrosis virus (AcMNPV) genome is circular dsDNA (~134 kb) and contains regions of highly repetitive DNA elements and genes in both strands [2]. This genomic DNA can be propagated in Escherichia coli as a bacmid, and genes can be inserted by Tn7 transposase based transposition into the virus DNA [3]
Summary
Many cellular multi-protein complexes are naturally present in cells at low abundance. One approach for the baculovirus mediated co-expression of multiple genes is the insertion of expression cassettes as tandem or inverted repeats at the polyhedrin locus in the viral genomic DNA [6,7,8,9,12,13]. This approach ensures that every cell in the culture expresses the genes encoding recombinant proteins in the same ratio and results in consistent yields of recombinant protein complex [14]. Before any multilocus approach to express genes for larger complexes could be pursued it would be necessary to characterise foreign gene expression at alternative loci within the AcMNPV genome
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