Show simple item record

dc.contributor.authorNoad, RJen_US
dc.contributor.authorStewart, Men_US
dc.contributor.authorBoyce, Men_US
dc.contributor.authorCelma, CCen_US
dc.contributor.authorWillison, KRen_US
dc.contributor.authorRoy, Pen_US
dc.identifier.citationBMC MOLECULAR BIOLOGY, 2009, 10en_US
dc.description.abstractBackground: 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 PI Crerecombinase 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/MI 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.en_US
dc.titleMultigene expression of protein complexes by iterative modification of genomic Bacmid DNAen_US
dc.typeJournal Article
rioxxterms.typeJournal Article/Reviewen_US
dc.relation.isPartOfBMC MOLECULAR BIOLOGYen_US
pubs.notesaffiliation: Roy, P (Reprint Author), Univ London London Sch Hyg & Trop Med, Dept Infect & Trop Dis, Keppel St, London WC1E 7HT, England. Noad, Rob J.; Stewart, Meredith; Boyce, Mark; Celma, Cristina C.; Roy, Polly, Univ London London Sch Hyg & Trop Med, Dept Infect & Trop Dis, London WC1E 7HT, England. Willison, Keith R., Inst Canc Res, Sect Cell & Mol Biol, London SW3 6JB, England. article-number: 87 keywords-plus: VIRUS-LIKE PARTICLES; NUCLEAR POLYHEDROSIS-VIRUS; AUTOGRAPHA-CALIFORNICA NUCLEOPOLYHEDROVIRUS; 4 STRUCTURAL PROTEINS; RECOMBINANT BACULOVIRUS; INSECT CELLS; ESCHERICHIA-COLI; BUDDED VIRUS; WILD-TYPE; HOMOLOGOUS RECOMBINATION research-areas: Biochemistry & Molecular Biology web-of-science-categories: Biochemistry & Molecular Biology author-email: orcid-numbers: Noad, Rob/0000-0002-0279-8461 funding-acknowledgement: Biotechnology and Biological Sciences Research Council, UK; Biotechnology and Biological Sciences Research Council [BB/C504735/1] funding-text: This work was supported by the Biotechnology and Biological Sciences Research Council, UK. The authors would like to gratefully acknowledge the gift of cDNA for influenza A SC35M and polyclonal antibody from H. D. Klenk ( University of Marburg), the E. coli strain EL350 from C. T. Dolphin ( King’s College, London), and the bacmid BAC10: KO1629 from I. M. Jones ( University of Reading). number-of-cited-references: 48 times-cited: 18 usage-count-last-180-days: 0 usage-count-since-2013: 6 journal-iso: BMC Mol. Biol. doc-delivery-number: 503QP unique-id: ISI:000270554500001 oa: gold da: 2018-08-29en_US
pubs.notesNot knownen_US
pubs.organisational-group/ICR/Primary Group
pubs.organisational-group/ICR/Primary Group/ICR Divisions
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Closed research teams
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Closed research teams/Chromatin Regulation
pubs.embargo.termsNot knownen_US
icr.researchteamChromatin Regulationen_US
dc.contributor.icrauthorWillison, Keithen_US

Files in this item


This item appears in the following Collection(s)

Show simple item record
Except where otherwise noted, this item's license is described as