title: Purification and reconstitution of human membrane-bound DHRS7 (SDR34C1) from Sf9 cells.
abstract: Dehydrogenase/reductase SDR family member 7 (DHRS7, SDR34C1, retSDR4) is one of the many endoplasmic reticulum bound members of the SDR superfamily. Preliminary results indicate its potential significance in human metabolism. DHRS7 containing TEV-cleavable His10 and FLAG-tag expressed in the Sf9 cell line was solubilised, purified, and reconstituted into liposomes to enable the improved characterisation of this enzyme in the future. Igepal CA-630 was determined to be the best detergent for the solubilisation process. The solubilised DHRS7 was purified using affinity chromatography, and the purified enzyme was subjected to TEV cleavage of the affinity tags and then repurified using subtractive Ni-IMAC. The cleaved and uncleaved versions of DHRS7 were successfully reconstituted into liposomes. In addition, using tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as the substrate, the cleaved liposomal DHRS7 was found to be inactive, whereas the pure and uncleaved liposomal DHRS7 were confirmed as enzymes, which reduce carbonyl group of the substrates.
authors: Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 50005 Hradec Kralove, Czech Republic.
authors: Adam
authors: A
authors: Skarka
authors: Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 50005 Hradec Kralove, Czech Republic.
authors: Lucie
authors: L
authors: Škarydová
authors: Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 50005 Hradec Kralove, Czech Republic.
authors: Hana
authors: H
authors: Štambergová
authors: Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 50005 Hradec Kralove, Czech Republic. Electronic address: wsol@faf.cuni.cz.
authors: Vladimír
authors: V
authors: Wsól
mesh_terms: D000818:Animals; D002462:Cell Membrane; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D008565:Membrane Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases
keywords: Carbonyl reductase activity; DHRS7; Purification; Reconstitution; Short-chain dehydrogenases/reductases; Solubilisation