Abstract 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 Escherichia coli 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 E. coli 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.

Kristina Shutevska

Aleksandra Kapedanovska Nestorovska

Acta Pharmaceutica

AP 是一本致力于制药和相关科学的国际多学科期刊，主要包含关于核心生物医学和健康主题的文章。 AP的目的是增加药物研究在学术界、工业界和实验室中的影响。 AP 非常强调质量和原创性，发布从药物发现到临床实践的报告。涵盖的主题有：分析、生物化学、生物制药、生物技术、细胞生物学、细胞培养、临床药学、药物设计、药物递送、药物处置、药物稳定性、基因技术、医学（包括诊断和治疗）、药物化学、新陈代谢、分子生物学建模、药理学（临床和动物）、肽和蛋白质化学、生药学、药物流行病学、药物经济学、药效学和药代动力学、蛋白质设计、放射性药物和毒理学。

Acta Pharmaceutica is an international multidisciplinary journal dedicated to the field of pharmaceuticals and related sciences, primarily publishing core articles on biomedical and health topics. The journal aims to enhance the impact of drug research in academia, industry, and laboratories. It places a strong emphasis on the quality and originality of research, publishing reports that cover the spectrum from drug discovery to clinical practice. Topics covered include: analysis, biochemistry, biopharmaceuticals, biotechnology, cell biology, cell culture, clinical pharmacy, drug design, drug delivery, drug disposition, drug stability, genetic technology, medicine (including diagnostics and therapeutics), medicinal chemistry, metabolism, molecular biology modeling, pharmacology (clinical and animal), peptide and protein chemistry, pharmacognosy, pharmacoepidemiology, pharmacoeconomics, pharmacodynamics and pharmacokinetics, protein design, radiopharmaceuticals, and toxicology.

《Acta Pharmaceutica》是一本国际多学科期刊，专注于制药及相关科学领域，主要发表关于生物医学和健康主题的核心文章。该期刊旨在提升药物研究在学术界、工业界和实验室中的影响力。期刊特别强调研究的质量和原创性，涵盖从药物发现到临床实践的各类报告。涉及的主题包括：分析、生物化学、生物制药、生物技术、细胞生物学、细胞培养、临床药学、药物设计、药物递送、药物处置、药物稳定性、基因技术、医学（包括诊断和治疗）、药物化学、新陈代谢、分子生物学建模、药理学（临床和动物）、肽和蛋白质化学、生药学、药物流行病学、药物经济学、药效学和药代动力学、蛋白质设计、放射性药物和毒理学。

Category	Quartile
医学	4
医学, 药学	4

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

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Journal Info

Category	Quartile
PHARMACOLOGY & PHARMACY	3