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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.

SUBMITTER: Lockard MA 

PROVIDER: S-EPMC3118733 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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A high-throughput immobilized bead screen for stable proteins and multi-protein complexes.

Lockard Meghan A MA   Listwan Pawel P   Pedelacq Jean-Denis JD   Cabantous Stéphanie S   Nguyen Hau B HB   Terwilliger Thomas C TC   Waldo Geoffrey S GS  

Protein engineering, design & selection : PEDS 20110603 7


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 be  ...[more]

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