title: ORF-selector ESPRIT: a second generation library screen for soluble protein expression employing precise open reading frame selection.
abstract: Here we present ORF-selector ESPRIT, a 9-fold enhanced version of our technology for screening incremental truncation libraries to identify soluble high yielding constructs of challenging proteins. Gene fragments are truncated at both termini to access internal domains and the resulting reading frame problem is addressed by an unbiased, intein-based open reading frame selection yielding only in-frame DNA inserts. This enriched library is then subcloned into a standard high-level expression plasmid where tens of thousands of constructs can be assayed in a two-step process using colony- and liquid-handling robots to isolate rare highly expressing clones useful for production of multi milligram quantities of purifiable proteins. The p85α protein was used to benchmark the system resulting in isolation of all known domains, either alone or in tandem. The human kinase IKK1 was then screened resulting in purification of a predicted internal domain. This strategy provides an integrated, facile route to produce soluble proteins from challenging and poorly understood target genes at quantities compatible with structural biology, screening applications and immunisation studies. The high genetic diversity that can be sampled opens the way to study more diverse systems including multisubunit complexes.
authors.affiliation: Grenoble Outstation, European Molecular Biology Laboratory, 6 rue Jules Horowitz, BP181, 38042 Grenoble Cedex 9, France.
authors.forename: Yingfeng
authors.initials: Y
authors.lastname: An
authors.forename: Hayretin
authors.initials: H
authors.lastname: Yumerefendi
authors.forename: Philippe J
authors.initials: PJ
authors.lastname: Mas
authors.forename: Alban
authors.initials: A
authors.lastname: Chesneau
authors.forename: Darren J
authors.initials: DJ
authors.lastname: Hart
mesh_terms: D057205:Automation, Laboratory; D058543:Class Ia Phosphatidylinositol 3-Kinase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D051550:I-kappa B Kinase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D016366:Open Reading Frames; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D012016:Reference Values; D012995:Solubility
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D058543:Class Ia Phosphatidylinositol 3-Kinase; C496559:CHUK protein, human; D051550:I-kappa B Kinase