Purification and reconstitution of human membrane-bound DHRS7 (SDR34C1) from Sf9 cells
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- 作者：Adam Skarka ; Lucie 艩karydov谩 ; Hana 艩tambergov谩 ; Vladim铆r Ws贸l
- 关键词：Short-chain dehydrogenases/reductases ; DHRS7 ; Solubilisation ; Purification ; Reconstitution ; Carbonyl reductase activity
- 刊名：Protein Expression and Purification
- 出版年：March, 2014
- 年：2014
- 卷：95
- 期：Complete
- 页码：44-49
- 全文大小：709 K
文摘
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.