Recombinant Mouse TDO2 His-tag Protein, CF Summary
with a C terminal 6-His tag
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.|
|Shipping||The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer: 50 mM MES, pH 6.5
- 0.405 M Tris, pH 8.0
- Recombinant Mouse TDO2 (rmTDO2) (Catalog # 10001-TD)
- Ascorbic Acid (Sigma, Catalog # 255564), 500 mM stock in deionized water
- L-Tryptophan (Sigma, Catalog # T0254), 40 mM stock in deionized water
- Catalase (Sigma, Catalog # C30), 100,000 Units/mL stock in Assay Buffer
- Methylene Blue (Sigma, Catalog # 28514), 10 mM stock in deionized water
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Prepare Substrate Mixtures.
a. Dilute L-Tryptophan to 8 mM in Assay Buffer
b. Dilute Ascorbic acid to 80 mM in 0.405 M Tris, pH 8.0.
c. Prepare a mixture of 9000 Units/mL catalase, and 40 µM Methylene Blue in Assay Buffer.
d. Mix equal volumes of 1b and 1c for final concentrations of 40 mM Ascorbic Acid, 4500 units/mL Catalase, and 20 µM Methylene Blue.
- Dilute rmTDO2 to 40 ng/µL in Assay Buffer.
- Load 25 µL of 40 ng/µL rmTDO2 to clear plate, and start the reaction by adding 25 µL of 8 mM L-Tryptophan followed by 50 µL of Mixture 1d. Include a Substrate Blank containing 25 µL Assay Buffer, 25 µL 8 mM L-Tryptophan and 50 µL of Mixture 1d.
- Read plate in kinetic mode for 5 minutes at an absorbance of 321 nm.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol|
|ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)|
**Using the extinction coefficient 3750 M-1cm-1.
***Using the path correction 0.32 cm.
Note: the output of many spectrophotometers is in mOD. Per Well:
- rmTDO2: 1.0 µg
- Ascorbic Acid: 20 mM
- L-Tryptophan: 2 mM
- Catalase: 225 units
- Methylene Blue: 10 µM
Recombinant Mouse TDO2 (Catalog # 10001-TD) is measured by its ability to oxidize L-tryptophan to N-formyl-kynurenine.
1 μg/lane of Recombinant Mouse TDO2 was resolved with SDS-PAGE underreducing (R) and non-reducing (NR) conditions and visualized by silver staining,showing bands at 39 kDa and 75 kDa under R conditions.
Tryptophan 2,3-dioxygenase (TDO2), a heme-containing cytosolic dioxygenase, forms a homo-tetrameric active molecule of approximately 190 kDa composed of 48 kDa monomers (1, 2). Mouse TDO2 shares 89% aa sequence identity with human TDO2. TDO2 is one of three proteins capable of catalyzing the first and rate-limiting step of the L-kynurenine pathway (KP): oxidative cleavage of the essential amino acid L-tryptophan to form N formyl kynurenine (3). TDO2 is a cytosolic protein typically localized to the liver and brain, unlike the more ubiquitously expressed indoleamine 2,3-dioxygenase (IDO), yet it is responsible for ~90% of the primary route of catabolism of tryptophan through the KP (3). TDO2 is upregulated in extrahepatic tumors (4-6) and is consequently a target in cancer immunotherapy (7). TDO2 is a therapeutic target in brain disease such as schizophrenia, Alzheimers disease, multiple sclerosis and glioma (8-11) due to its role in the regulation of levels of critical biologically active downstream KP metabolites (3). Polymorphisms in the TDO2 gene have been implicated for a role in behavioural responses and autism (12,13).
- Lewis-Ballester, A. et al. (2016) Sci. Rep. 6:35169.
- Rafice, S.A. et al. (2009) Biochem. Soc. Trans. 37:408.
- Badawy, A. (2017) Int J. Tryptophan Res. 10:1.
- Pilotte, L. et al. (2012) Proc. Natl. Acad. Sci. U.S.A. 109:2497.
- D'Amato, N.C. et al. (2015) Cancer Res. 75:4651.
- Yu, C.P et al. (2017) Med. Oncol. 34:73.
- Platten, et al. (2015) Front. Immunol. 5:673.
- Breda, C. et al. (2016) Proc. Natl. Acad. Sci. U.S.A. 113:5435
- Yu, C.P. et al. (2016) Metab. Brain Dis. 31:737.
- Lanz. T.V. et al. (2017) Sci. Rep. 7:41271.
- Reus, G.Z. et al. (2018) Prog. Neuropsychopharmacol. Biol. Psychiatry 81:55.
- Soichot, M. et al. (2013) Alcohol Alcohol 48:415.
- Nabi, R. et al. (2004) Am. J. Med. Genet. B. Neuropsychiatr. Genet. 125B:63.
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