Recombinant Human D-Amino Acid Oxidase His-tag Protein, CF
Recombinant Human D-Amino Acid Oxidase His-tag Protein, CF Summary
with N-terminal Met and 6-His tag
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|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.
- Buffer: 50 mM Sodium Phosphate, pH 8.0
- Flavin Adenine Dinucleotide (FAD) (Sigma, Catalog # F6625), 10 mM stock in deionized water
- Recombinant Human D-Amino Acid Oxidase His-tag (rhDAO) (Catalog # 10116-DO)
- Coupling Enzyme: Horseradish Peroxidase (HRP) (250-333 U/mg) (Sigma, Catalog # P8375), 250 units/mL stock in 0.1 M Sodium Phosphate, pH 8.0
- Substrate Component 1: D-Alanine (Sigma, Catalog # A7377), 500 mM stock in deionized water
- Substrate Component 2: Amplex Ultra Red (AUR) (Molecular Probes, Catalog # A36006), 10 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Prepare Assay Buffer (50 mM Sodium phosphate, 5 µM FAD, pH 8.0) by adding 10 mM stock of FAD to Buffer right before use.
- Dilute rhDAO to 2 ng/µL in Assay Buffer.
- Dilute D-Alanine to 40 mM in Assay Buffer.
- Combine equal volumes of 2 ng/µL rhDAO and 40 mM D-Alanine. Also, create a Substrate Blank by combining equal volumes of Assay Buffer and 40 mM D-Alanine.
- Incubate the reactions for 10 minutes at 37 °C.
- Prepare Substrate Mixture containing 2 units/mL HRP and 100 µM AUR in Assay Buffer.
- Load 50 µL of the incubated reactions into the wells of a black well plate, and start the reaction by adding 50 µL of Substrate Mixture.
- Read at excitation and emission wavelengths of 544 nm and 590 nm (top read), respectively in endpoint mode. Note: A cutoff must be set manually at a wavelength of 570 nm.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU)|
|Incubation time (min) x amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Derived using a fluorescent standard prepared by incubating 50 µM AUR, 1 unit/mL HRP, 10 mM D-Alanine, and a curve of Hydrogen Peroxide (Sigma, Catalog # H1009) in Assay Buffer. Use this oxidized AUR curve to determine the conversion factor.
- rhDAO: 0.05 µg
- D-Alanine: 10 mM
- HRP: 1 unit/mL
- AUR: 50 µM
1 μg/lane of Recombinant Human D-Amino Acid Oxidase His-tag (Catalog # 10116-DO) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by silver staining, showing a band at 39 kDa.
Background: D-Amino Acid Oxidase
D-amino acid oxidase (DAO) is an FAD-dependent enzyme that catalyzes the strict stereoselective oxidative deamination of D-amino acids. Human DAO is an 80 kDa stable homodimer of monomers in head to head conformation (1). Each monomer has two domains: a substrate-binding domain containing the active site and responsible for oligomerization and the FAD-binding domain (2). Mammalian DAO is primarily expressed in kidney, liver and brain, but also in small intestine and neutrophilic leukocytes (3). Human DAO differs in biophysical characteristics compared to other species due to its weakly bound cofactor, modulation by the regulatory protein pLG72 (4), and relatively low activity on its physiological substrates: D-serine, D-Cys, and D-DOPA (5). D-serine is a key neuromodulator and co-agonist of N-methyl-D-aspartate receptors (NMDAR), receptors that are critically involved in learning and memory. Abnormal levels of D-serine are associated with chronic neurodegeneration and misregulation of DAO. While many variants of DAO are implicated in neurological disease, a specific DAO gene mutation resulting in an enzyme with impaired activity causes dominantly inherited familial amyotrophic lateral sclerosis (ALS) (6). Only variants in the DAO gene are significantly associated with the clinical outcome of ALS patients (7). In Alzheimer's disease (AD), DAO expression is higher and correlates with severity of cognitive impairment (8) while inhibition resulted in improved cognitive function in AD patients (9). Likewise, as with AD, increased expression of DAO with concomitant decreased D-serine levels is found in schizophrenia with beneficial results observed upon therapeutic delivery of D-serine (10). DAO inhibition has also been shown to modulate chronic pain (11, 12). DAO is a pharmaceutical target for the treatment of neurological therapeutic disease and pain (13, 14).
- Molla, G. et al. (2006) FEBS Lett. 580:2358.
- Kawazoe, T. et al. (2006) Protein Sci. 15:2708.
- Sacchi, S. et al. (2018) Front. Mol. Biosci. 5:55.
- Sacchi, S. et al. (2008) J. Biol. Chem. 283:22244.
- Kawazoe, T. et al. (2007) Biochem. Biophys. Res. Commun. 355:385.
- Mitchell, J. et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107:7556.
- Cirulli, E.T. et al. (2015) Science 347:1436.
- Lin, C. H. et al. (2017) J. Alzhemier's Dis. 56:959.
- Lin, C.H. et al. (2014) Curr. Pharm. Des. 20:5169.
- Heresco-Levy, U. et al. (2005) Biol. Psychiatry 57:577.
- Zhou, W.J. et al. (2010) J. Pharmacol. Exp. Ther. 332:248.
- Gong, N. et al. (2011) J. Pharmacol. Exp. Ther. 336:282.
- Szilagyi, B. et al. (2018) Expert Opin. Drug Discov. 13:973.
- Khangura, R.K. et al. (2019) Korean J. Physiol. Pharmacol. 23:1.
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