title: Expression in Escherichia coli of the catalytic domain of human proline oxidase.
abstract: The human PRODH gene has been shown to have unique roles in regulating cell survival and apoptotic pathways and it has been related to velocardiofacial syndrome/DiGeorge syndrome and increased susceptibility to schizophrenia. It encodes for the flavoprotein proline oxidase (PO), which catalyzes the conversion of l-proline to Δ(1)-pyrroline-5-carboxylate. Despite the important physiological and medical interest in human PO, up to now only microbial homologues of PO have been expressed as recombinant protein and fully characterized. By using a bioinformatics analysis aimed at identifying the catalytic domain and the regions with a high intrinsic propensity to structural disorder, we designed deletion variants of human PO that were successfully expressed in Escherichia coli as soluble proteins in fairly high amounts (up to 10mg/L of fermentation broth). The His-tagged PO-barrelN protein was isolated as an active (the specific activity is 0.032U/mg protein), dimeric holoenzyme showing the typical spectral properties of FAD-containing flavoprotein oxidases. These results pave the way for elucidating structure-function relationships of this human flavoenzyme and clarifying the effect of the reported polymorphisms associated with disease states.
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell'Insubria, Varese, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Tallarita
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
authors.forename: Stefano
authors.initials: S
authors.lastname: Servi
authors.forename: Gianluca
authors.initials: G
authors.lastname: Molla
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011394:Proline Oxidase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility
chemical_list: D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011394:Proline Oxidase