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

Journal Article · · Protein Engineering, Design and Selection
 [1];  [1];  [2];  [3];  [1];  [1];  [1]
  1. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
  2. Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Centre National de la Recherche Scientifique (CNRS), Toulouse (France). Institut de Pharmacologie et Biologie Structurale (IPBS); Universite de Toulouse (France). UPS
  3. Los Alamos National Lab. (LANL), Los Alamos, NM (United States); INSERM, Toulouse (France); Universite de Toulouse (France); Institut Claudius Regaud, Toulouse (France)
+ Show Author Affiliations

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 Talonw 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.

Research Organization:
Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division
Grant/Contract Number:
AC52-06NA25396
OSTI ID:
1625584
Journal Information:
Protein Engineering, Design and Selection, Vol. 24, Issue 7; ISSN 1741-0126
Publisher:
Oxford University PressCopyright Statement
Country of Publication:
United States
Language:
English

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Cited By (3)

Hot CoFi Blot: A High-Throughput Colony-Based Screen for Identifying More Thermally Stable Protein Variants book January 2019
Beyond the ribosome: proteome-wide secretability studies with SECRiFY posted_content January 2018
A New Protein-Protein Interaction Sensor Based on Tripartite Split-GFP Association journal October 2013

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59 BASIC BIOLOGICAL SCIENCES
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