title: 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.affiliation: Grenoble Outstation, European Molecular Biology Laboratory, BP181 Grenoble, France.
authors.forename: Yingfeng
authors.initials: Y
authors.lastname: An
authors.forename: Patrick
authors.initials: P
authors.lastname: Meresse
authors.forename: Philippe J
authors.initials: PJ
authors.lastname: Mas
authors.forename: Darren J
authors.initials: DJ
authors.lastname: Hart
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D015870:Gene Expression / Q000502:physiology; D015723:Gene Library; D053263:Gene Regulatory Networks; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D040081:Protein Array Analysis / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012107:Research Design; D012995:Solubility
chemical_list: D046912:Multiprotein Complexes; D011994:Recombinant Proteins