• Recombinant expression of DT-Diaphorase enzyme: A new approach for activation of cancer chemotherapeutic prodrugs
  • hamideh askari,1 faeze khakbaz,2 maryam zaboli,3 masoud torkzade-mahani,4,*
    1. Department of Biotechnology, Institute of Science, High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran
    2. Department of Nano Chemistry, Faculty of Chemistry, Shahid Bahonar University, Kerman, Iran.
    3. department of chemistry, faculty of science, university of Birjand, Birjand, Iran
    4. Department of Biotechnology, Institute of Science, High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran


  • Introduction: DT-Diaphorase (flavin-containing enzyme) as a member of oxidoreductase family has been shown to have a role in activation of quinone-containing cancer chemotherapeutic prodrugs to their active form, as well as inactivation of a variety of xenobiotics involved in carcinogenesis and also has received wide attention as the key anodic enzyme mediating the electron transfer and electric energy generation in enzymatic biofuel cells (EBFCs). According to significant roles of this enzyme in many fields of biotechnology specially in cancer studies, biosensor design and in vitro diagnostic tests, we describe optimum recombinant expression conditions, characterization and application of a DT-Diaphorase enzyme.
  • Methods: The transformed recombinant plasmids to E. coli BL21 competent cells were used for protein expression. Recombinant histidine -tailed DTD enzyme was expressed in 10 mL LB medium containing 80 μ g/m L kanamycin at 37oC for 16 hours. Then the cells were collected by centrifugation at 1500 g for 10 minutes at 4oC and suspended in 4 mL of LB medium. The seeding was added to Terrific Broth (TB) medium to cell cultured reached an OD600 of about 0.1 and were incubated at 37oC with shaking at 180 rpm. When the cell cultures reached an OD600 0f 0.6, cells were incubated with IPTG (1mL, 1mM) in various temperatures (18, 25, 30 and 37oC) and times (5 and 10 hours). The bacterial cells were collected by centrifugation at 6000 rpm for 10 minutes at 4oC and were suspended in detergent lysis buffer. Several cycles sonication in ice bath was used for broken the bacterial cells walls. The supernatant was collected using centrifuge at 13000 rpm for 20 minutes at 4oC. The Ni-sepharose affinity and size exclusion chromatography were used for purification of prepared DTD enzyme. SDS –PAGE analysis, activity measurement (according to the absorption of formazan as a product of enzymatic reaction at 550 nm using cary 50 UV-Vis spectrophotometer) and Bradford assay were used for study the enzyme properties.
  • Results: To obtain the best protein expression condition, two of the most important effective factors (incubation time and temperature) were optimized. The highest expression level of protein expression was examined by various factors such as single and broad 47 kDa band in SDS-PAGE analysis, high activity, and high enzyme concentration. According to obtained results, incubation of bacterial cells in 18oC for 5 hours can be the best condition for DTD expression.
  • Conclusion: DT-Diaphorase as an important enzyme in biotechnology such as early detection of cancer has attracted much attention. In this work, the optimized condition for DTD expression have been reported (incubation in 18oC for 5 hours). According to a colorimetric rout as a sensitive method for investigation the activity of prepared enzyme, the obtained DTD in the optimal conditions shows the activity as much as 10.76 units per 5 μL. A single and sharp 47kDa band in SDS-PAGE analysis is the other evidence for success enzyme expression.
  • Keywords: Enzyme expression, DT-Diaphorase