Purification and reconstitution of human membrane-bound DHRS7 (SDR34C1) from Sf9 cells
Language English Country United States Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
24316191
DOI
10.1016/j.pep.2013.11.013
PII: S1046-5928(13)00257-X
Knihovny.cz E-resources
- Keywords
- Carbonyl reductase activity, DHRS7, Purification, Reconstitution, Short-chain dehydrogenases/reductases, Solubilisation,
- MeSH
- Cell Membrane MeSH
- Humans MeSH
- Membrane Proteins chemistry genetics isolation & purification metabolism MeSH
- Oxidoreductases chemistry genetics isolation & purification metabolism MeSH
- Recombinant Proteins chemistry genetics isolation & purification metabolism MeSH
- Sf9 Cells MeSH
- Spodoptera MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Membrane Proteins MeSH
- Oxidoreductases MeSH
- Recombinant Proteins MeSH
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.
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