title: Production of unstable proteins through the formation of stable core complexes.
abstract: Purification of proteins that participate in large transient complexes is impeded by low amounts, heterogeneity, instability and poor solubility. To circumvent these difficulties we set up a methodology that enables the production of stable complexes for structural and functional studies. This procedure is benchmarked and applied to two challenging protein families: the human steroid nuclear receptors (SNR) and the HIV-1 pre-integration complex. In the context of transcriptional regulation studies, we produce and characterize the ligand-binding domains of the glucocorticoid nuclear receptor and the oestrogen receptor beta in complex with a TIF2 (transcriptional intermediary factor 2) domain containing the three SNR-binding motifs. In the context of retroviral integration, we demonstrate the stabilization of the HIV-1 integrase by formation of complexes with partner proteins and DNA. This procedure provides a powerful research tool for structural and functional studies of proteins participating in non-covalent macromolecular complexes.
authors.affiliation: IGBMC, UDS, CNRS, INSERM, 1 rue Laurent Fries, 67404 Illkirch, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Levy
authors.affiliation: IGBMC, UDS, CNRS, INSERM, 1 rue Laurent Fries, 67404 Illkirch, France.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Eiler
authors.affiliation: IGBMC, UDS, CNRS, INSERM, 1 rue Laurent Fries, 67404 Illkirch, France.
authors.forename: Karine
authors.initials: K
authors.lastname: Pradeau-Aubreton
authors.affiliation: IGBMC, UDS, CNRS, INSERM, 1 rue Laurent Fries, 67404 Illkirch, France.
authors.forename: Benoit
authors.initials: B
authors.lastname: Maillot
authors.affiliation: IGBMC, UDS, CNRS, INSERM, 1 rue Laurent Fries, 67404 Illkirch, France.
authors.forename: François
authors.initials: F
authors.lastname: Stricher
authors.affiliation: IGBMC, UDS, CNRS, INSERM, 1 rue Laurent Fries, 67404 Illkirch, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Ruff
mesh_terms: D002460:Cell Line; D015497:HIV-1 / Q000378:metabolism; D006801:Humans; D046912:Multiprotein Complexes / Q000302:isolation & purification / Q000378:metabolism*; D055550:Protein Stability; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D012995:Solubility; D012997:Solvents
chemical_list: D046912:Multiprotein Complexes; D018160:Receptors, Cytoplasmic and Nuclear; D012997:Solvents