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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
2
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pubmed:dateCreated |
2011-3-2
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pubmed:abstractText |
Structural and biophysical studies of protein complexes require multi-milligram quantities of soluble material. Subunits are often unstable when expressed separately so co-expression strategies are commonly employed since in vivo complex formation can provide stabilising effects. Defining constructs for subunit co-expression experiments is difficult if the proteins are poorly understood. Even more problematic is when subunit polypeptide chains co-fold since individually they do not have predictable domains. We have developed CoESPRIT, a modified version of the ESPRIT random library construct screen used previously on single proteins, to express soluble protein complexes. A random library of target constructs is screened against a fixed bait protein to identify stable complexes. In a proof-of-principle study, C-terminal fragments of the influenza polymerase PB2 subunit containing folded domains were isolated using importin alpha as bait. Separately, a C-terminal fragment of the PB1 subunit was used as bait to trap N-terminal fragments of PB2 resulting in co-folded complexes. Subsequent expression of the target protein without the bait indicates whether the target is independently stable, or co-folds with its partner. This highly automated method provides an efficient strategy for obtaining recombinant protein complexes at yields compatible with structural, biophysical and functional studies.
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pubmed:commentsCorrections |
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:issn |
1932-6203
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pubmed:author |
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pubmed:issnType |
Electronic
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pubmed:volume |
6
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
e16261
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pubmed:meshHeading |
pubmed-meshheading:21364980-Amino Acid Sequence,
pubmed-meshheading:21364980-Animals,
pubmed-meshheading:21364980-Cloning, Molecular,
pubmed-meshheading:21364980-Gene Expression,
pubmed-meshheading:21364980-Gene Library,
pubmed-meshheading:21364980-Gene Regulatory Networks,
pubmed-meshheading:21364980-High-Throughput Screening Assays,
pubmed-meshheading:21364980-Humans,
pubmed-meshheading:21364980-Models, Biological,
pubmed-meshheading:21364980-Models, Molecular,
pubmed-meshheading:21364980-Multiprotein Complexes,
pubmed-meshheading:21364980-Plasmids,
pubmed-meshheading:21364980-Protein Array Analysis,
pubmed-meshheading:21364980-Recombinant Proteins,
pubmed-meshheading:21364980-Research Design,
pubmed-meshheading:21364980-Solubility
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pubmed:year |
2011
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pubmed:articleTitle |
CoESPRIT: a library-based construct screening method for identification and expression of soluble protein complexes.
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pubmed:affiliation |
Grenoble Outstation, European Molecular Biology Laboratory, BP181 Grenoble, France.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't,
Validation Studies
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