| Assay Method Information | |
| | TREX1 Exonuclease Assay |
| Description: | For human TREX1, the nucleotide sequence of the gene construct was codon optimized for expression in the bacterial host. The sequence was incorporated into the pMAL-c5e vector (NEB) downstream of a solubility-promoting MBP (maltose binding protein) fusion partner. The fusion protein product was expressed in E. coli strain BL21 (DE3) (Millipore) and purified in a series of chromatographic steps. Initial steps were conducted using a dextrin sepharose affinity column followed by a Q-sepharose ion exchange column (both GE Healthcare). After the second column the MBP partner was removed by incubation with enterokinase (NEB). Further purification was carried out with the second application of a Q-sepharose column followed by a Superdex 75 (GE Healthcare) size exclusion column. Finally, a heparin sepharose column (GE Healthcare) was applied to remove contaminating nucleotides. Similar methods were used in the preparation of the murine TREX1 enzyme and are described in Example B set forth below.To evaluate the effect of compounds on TREX1 activity, test compounds were serially diluted (11-point, 3-fold) from 10 mM stock solutions and delivered to 384-well low-volume assay plates in 80 nL DMSO using an acoustic dispenser. Next, 4 μL of human TREX1 (0.5 nM), or murine TREX1 (1 nM), diluted in assay buffer (20 mM Tris pH 7.5, 5 mM MgCl, 100 μg/mL BSA, 0.002% Triton X-100, 2 mM DTT), was added to the assay plate. After incubating for 30 minutes, 4 μL of labeled DNA oligonucleotide (500 nM) in assay buffer was added to initiate TREX1 exonuclease activity. The reaction was allowed to proceed for 45 minutes at room temperature prior to the addition of 4 μL of 150 mM EDTA to halt TREX1 activity. Assay plates were equilibrated for an additional 30 minutes and read on an EnVision Plate Reader (Perkin Elmer) to measure fluorescence emission at 535 nm following excitation at 485 nm. Fluorescence was plotted as a function of log molar compound concentration and fit to a four-parameter dose-response equation to determine compound IC50. |
| Affinity data for this assay | |
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