One step affinity recovery of 3α-hydroxysteroid dehydrogenase from cloned Escherichia coli

• 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 amino-Sepharose with the aid of the spacers.
• After one affinity chromatography step only, 3α-HSD recovering sample with purity of 94% was obtained.
• 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 ∼56 kDa by 15.0% SDS-PAGE analysis and SE-HPLC analysis.

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 ∼56 kDa 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 4.5 μg/g medium and 21.3 mg/g medium, respectively.