title: A high-throughput immobilized bead screen for stable proteins and multi-protein complexes.
abstract: We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1-10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon(®) resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length 'breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit.
authors.affiliation: Biosciences Division, MS-M888, Los Alamos National Laboratory, PO Box 1663, Los Alamos, NM 87545, USA.
authors.forename: Meghan A
authors.initials: MA
authors.lastname: Lockard
authors.forename: Pawel
authors.initials: P
authors.lastname: Listwan
authors.forename: Jean-Denis
authors.initials: JD
authors.lastname: Pedelacq
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Cabantous
authors.forename: Hau B
authors.initials: HB
authors.lastname: Nguyen
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Terwilliger
authors.forename: Geoffrey S
authors.initials: GS
authors.lastname: Waldo
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000295:instrumentation*; D006639:Histidine / Q000032:analysis / Q000235:genetics; D006801:Humans; D046912:Multiprotein Complexes / Q000032:analysis* / Q000235:genetics; D020102:Mycobacterium smegmatis / Q000201:enzymology / Q000235:genetics; D009842:Oligopeptides / Q000032:analysis / Q000235:genetics; D055550:Protein Stability; D041543:Pyrobaculum / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics; D012995:Solubility
chemical_list: C471213:His-His-His-His-His-His; D046912:Multiprotein Complexes; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006639:Histidine