title: Towards eukaryotic structural complexomics.
abstract: Many eukaryotic proteins exist in large multisubunit assemblies and often show compromised folding or activity when their interaction partners are not present. Protein complexes in eukaryotes can contain ten or more subunits with individual polypeptides ranging in size up to several hundred kilodalton, severely restricting the application of conventional cloning strategies and imposing constraints on the choice of the expression host. Modern structural molecular biology often depends on introducing diversity into the specimens under investigation, including mutation, truncation and placement of purification aids. Current recombinant expression methods often require a disproportionate labor investment prior to multiprotein expression, and subsequent to expression and analysis do not provide for rapid revision of the experiment. We have developed reagents and protocols for rapid and flexible multiprotein complex expressions suitable for structural biology, focusing on multigene baculoviral vectors and their recombination mediated assembly. A top priority in protein science is automation. Our strategy can be readily adapted in a robotics setup, for baculovirus/insect cell expression of protein complexes, but likewise also for mammalian or prokaryotic hosts.
authors.affiliation: Institute for Molecular Biology and Biophysics, ETH Hönggerberg, 8093, Zurich, Switzerland.
authors.forename: Christoph
authors.initials: C
authors.lastname: Bieniossek
authors.forename: Imre
authors.initials: I
authors.lastname: Berger
mesh_terms: D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D005057:Eukaryotic Cells / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D018411:Spodoptera
chemical_list: D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins