One step affinity recovery of 3α-hydroxysteroid dehydrogenase from cloned Escherichia coli.
3α-Hydroxysteroid dehydrogenase (3α-HSD), from Comamonas Testosterone, catalyze reversibly the oxidoreduction of 3α-hydroxyl groups of the steroid hormones. The gene encoding 3α-HSD (hsdA) from Comamonas Testosterone was expressed in Escherchia coli BL21 (DE3). A protocol for recovering 3α-HSD based on affinity strategy was designed and employed. Deoxycholic acid was chosen as the affinity ligand, and it was linked to Sepharose 4B with the aid of the spacers as cyanuric chloride and ethanediamine. With this specific affinity medium, the enzyme recovery process consisted of only one chromatography step to capture 3α-HSD. The target protein, analyzed on HPLC Agilent SEC-5 column, was of 94% pure among the captured protein, and 98% with SDS-PAGE analysis. The yield of the expressed enzyme was 8.8% of crude extracted proteins; the recovery yield of 3α-HSD was 73.2%. 3α-HSD was revealed as a non-covalent homodimer with molecular mass of ∼56kDa by 15.0% SDS-PAGE analysis and SE-HPLC analysis. The desorption constant Kd and the theoretical maximum absorption Qmax on the affinity medium were 4.5μg/g medium and 21.3mg/g medium, respectively.
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China. Electronic address: 19891996@sina.com.
Hailin
H
Yang
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Yanan
Y
Fang
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Zhizhen
Z
Wang
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Ling
L
Zhang
D000327:Adsorption; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods; D003001:Cloning, Molecular; D020604:Comamonas testosteroni / Q000201:enzymology* / Q000235:genetics; D003840:Deoxycholic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006913:Hydroxysteroid Dehydrogenases / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012685:Sepharose / Q000737:chemistry
D011994:Recombinant Proteins; D003840:Deoxycholic Acid; D012685:Sepharose; D006913:Hydroxysteroid Dehydrogenases
3α-Hydroxysteroid dehydrogenase; Affinity recovery; E. coli; Ligand; Purification