CoESPRIT: a library-based construct screening method for identification and expression of soluble protein complexes.

Abstract

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.

• Authors: Yingfeng An, Patrick Meresse, Philippe J Mas, Darren J Hart
• Journal: PloS one (PLoS One) Vol. 6 Issue 2 Pg. e16261 (Feb 22 2011) ISSN: 1932-6203 [Electronic] United States
• PMID: 21364980 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Validation Study)

Chemical References

• Multiprotein Complexes
• Recombinant Proteins

Topics

• Amino Acid Sequence
• Animals
• Cloning, Molecular (methods)
• Gene Expression (physiology)
• Gene Library
• Gene Regulatory Networks
• High-Throughput Screening Assays (methods)
• Humans
• Models, Biological
• Models, Molecular
• Multiprotein Complexes (chemistry, genetics, isolation & purification, metabolism)
• Plasmids (genetics)
• Protein Array Analysis (methods)
• Recombinant Proteins (chemistry, genetics, metabolism)
• Research Design
• Solubility