Cloning, expression, and purification of recombinant AKR1D1 for therapeutic applications.

AKR1D1, a key enzyme in the aldo-keto reductase superfamily, plays a dual role in both steroid metabolism and bile acid synthesis by catalyzing the NADPH-dependent reduction of carbon-carbon double bonds, specifically converting 3-ketosteroid hormones into 5β-steroids. Positioned at the critical intersection of steroid hormone and bile acid metabolism, AKR1D1 has the potential to profoundly influence metabolic homeostasis and drug metabolism. Despite its importance, the enzyme's therapeutic implications and role in drug metabolism remain underexplored. This study presents an optimized methodology for the cloning, expression, and purification of AKR1D1 using an expression system. We identified optimal conditions for ligation and precise DNA sequencing, emphasizing the need for lower DNA concentrations and higher purity. Protein expression was evaluated in strains BL21 and Rosetta, with the highest yields achieved under extended incubation at 25 °C with controlled IPTG concentrations. Using freshly transformed cells was essential for maintaining consistent protein expression. The enzyme's activity was confirmed using a spectrofluorometric assay, demonstrating efficient reduction of testosterone to 5β-DHT. This optimized methodology facilitates the production of AKR1D1 with high specific activity, establishing a valuable platform for future research. It enables a deeper investigation into AKR1D1's contributions to drug metabolism and its therapeutic potential.