title: Definition and expression in E. coli of large fragments from the human lipid kinase phosphatidylinositol 4-kinase type III alpha, and purification of a 1100-residue N-terminal module.
abstract: The eukaryotic lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KA in higher eukaryotes) is a ubiquitous enzyme that synthesizes the plasma membrane pool of phosphatidylinositol 4-phosphate. This important phosphoinositide has key roles in different signalization pathways, vesicular traffic and cellular compartment identity. Moreover, human PI4K4A is an essential factor for hepatitis C virus replication. PI4KA is a large protein (2102 residues for human PI4KA) with the kinase domain making up the ca 400 C-terminal residues. There is essentially no structural information about the 1500N-terminal residues and no clue as to the function of most of this region of PI4KA. In this report, we use computational methods in order to delineate fragments of human PI4KA amenable to soluble production in Escherichia coli. We clone and express these fragments as GST-fusions and evaluate the soluble fraction of each protein. Finally, we produce and purify to homogeneity a 1100-residue PI4KA N-terminal fragment. Our results further suggest that PI4KA can be described as a two-module protein. They open the way to structural characterization of the N-terminal regulatory module of PI4KA.
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, CEA Saclay, 91191 Gif-sur-Yvette cedex, France.
authors.forename: Cyntia
authors.initials: C
authors.lastname: Taveneau
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, CEA Saclay, 91191 Gif-sur-Yvette cedex, France.
authors.forename: Karine
authors.initials: K
authors.lastname: Blondeau
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, CEA Saclay, 91191 Gif-sur-Yvette cedex, France. Electronic address: stephane.bressanelli@i2bc.paris-saclay.fr.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Bressanelli
mesh_terms: D019870:1-Phosphatidylinositol 4-Kinase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019295:Computational Biology; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D011993:Recombinant Fusion Proteins; D019870:1-Phosphatidylinositol 4-Kinase
keywords: Bio-informatics; PI4KA regulation; Phosphatidylinositol 4-kinase; Production in E. coli of large recombinant proteins