title: Recombinant expression and reconstitution of multiprotein complexes by the USER cloning method in the insect cell-baculovirus expression system.
abstract: The capacity to reconstitute complex biological processes in vitro is a crucial step in providing a quantitative understanding of these systems. It provides material for structural, biochemical and biophysical analyses and allows the testing of biological hypotheses and the introduction of chemical probes and tags for single molecule analysis. Reconstitution of these systems requires access to homogenous components, usually through their over-production in heterologous over-expression systems. Here we describe the application of the USER (Uracil-Specific Excision Reagent) ligation-free cloning method to assemble recombinant MultiBac transfer vectors for the generation of recombinant baculovirus suitable for the expression of multi-protein complexes in insect cells.
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
authors.forename: Ziguo
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom. Electronic address: dbarford@mrc-lmb.cam.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Barford
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry* / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera; D019076:Transgenes; D014498:Uracil / Q000737:chemistry / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D014498:Uracil
keywords: Baculovirus insect cell expression; Multi-protein complexes; MultiBac; USER ligation-free cloning