Citation
- Authors: Park MG. et al.
- Year: 2023
- Journal: J Enzyme Inhib Med Chem 38 309-318
- Applications: in vitro / DNA / FectoPRO
- Cell type: Expi293F
Description: Human embryonic kidney Fibroblast
Known as: Expi 293-F, Expi, HEK-293 Expi
Method
The recombinant human ODC was overexpressed using Expi293FTM expression system (Thermo Fisher Scientific, A14635). Just prior to transient transfection, 5.4 108 cells of Expi293FTM were added to 180 mL of Expi293TM Expression Medium (3 106 cells/mL) in a 1-L flask with vent cap (Corning, CC-431147) incubated at 125 rpm, 8% CO2, 37 C. For 200-mL transfection in each 1-L flask, the plasmid DNA encoding C-terminal Twin-Strep-tagged recombinant human ODC was transfected into Expi293FTM cells using FectoPRO transfection kit (Polyplus) according to manufacturer’s protocol. Then, enhancer was added immediately. After 24 h, the incubation temperature was lowered from 37 C to 32 C to slow cell growth rate while further enhancing protein expression yield36,38. Overexpression of recombinant human ODC1 was monitored for 24 to 48 h via IRES-driven GFP expression in Expi293TM cells until transfection efficiency was reached to more than 60%. Lastly, the transfected cells were harvested and kept at 80 C for long-term storage.
Abstract
Ornithine decarboxylase (ODC), the first rate-limiting enzyme in polyamine synthesis, has emerged as a therapeutic target for cancer and Alzheimer's disease (AD). To inhibit ODC, α-difluoromethylornithine (DFMO), an irreversible ODC inhibitor, has been widely used. However, due to its poor pharmacokinetics, the need for discovery of better ODC inhibitors is inevitable. For high-throughput screening (HTS) of ODC inhibitors, an ODC enzyme assay using supramolecular tandem assay has been introduced. Nevertheless, there has been no study utilising the ODC tandem assay for HTS, possibly due to its intolerability to dimethyl sulfoxide (DMSO), a common amphipathic solvent used for drug libraries. Here we report a DMSO-tolerant ODC tandem assay in which DMSO-dependent fluorescence quenching becomes negligible by separating enzyme reaction and putrescine detection. Furthermore, we optimised human cell-line-based mass production of ODC for HTS. Our newly developed assay can be a crucial first step in discovering more effective ODC modulators than DFMO.