Expression in Escherichia coli of the catalytic domain of human proline oxidase

Protein Expression and Purification
Elena TallaritaGianluca Molla

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 dise...Continue Reading

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Citations

Jul 17, 2012·Free Radical Biology & Medicine·Sathish Kumar NatarajanDonald F Becker
Sep 4, 2014·Redox Biology·Renata L S GoncalvesMartin D Brand
Jun 9, 2016·Molecular Plant-microbe Interactions : MPMI·Georgina FabroMaría Elena Alvarez
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Apr 13, 2018·Biochemistry·John J TannerDonald F Becker
Sep 21, 2021·ACS Chemical Biology·Ashley C CampbellJohn J Tanner

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