Recombinant Human Kynureninase Protein, CF Summary
Met1-Asn465, with a C-terminal 10-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 MES and NaCl.|
|Shipping||The product is shipped with polar packs. 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 Tris, 0.05% (w/v) Brij-35, 5 µM Pyridoxal Phosphate (Sigma, Catalog # P9255), pH 8.0
- Recombinant Human Kynureninase (rhKYNU) (Catalog # 4887-KH)
- Substrate: 3-hydroxy-DL-kynurenine (3-HK) (Sigma, Cat. # H1771), 10 mM in 50% DMSO, 50% deionized water
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhKYNU to 2 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- Load in a black well plate 50 µL of 2 ng/µL rhKYNU, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 μL Assay Buffer and 50 µL 200 µM Substrate.
- Read at excitation and emission wavelengths of 315 nm and 415 nm (top read), respectively, in kinetic mode for 5 minutes.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)|
|amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Derived using calibration standard 3-hydroxyanthranilinic acid (Sigma, Catalog # H9391).
- rhKYNU: 0.1 µg
- Substrate: 100 µM
Kynureninase is a pyridoxal-5 (-phosphate-dependent enzyme that catalyzes the hydrolytic cleavage of the amino acids L-kynurenine and L-3-hydroxykynurenine to give either anthranilic acid or 3-hydroxyanthranilic acid and alanine (1). The enzyme is a member of the “kynurenine pathway” enzymes, through which the majority of dietary tryptophan is degraded in the liver, and is involved in the de novo biosynthesis of NAD+ (2, 3). Kynurenine pathway genes are expressed in immune system cells such as macrophages and microglia. During inflammatory responses, the kynurenine pathway in these cells produces quinolinic acid (QA) and not NAD+. QA excites neurons via the activation of NMDA (N-methyl-D-aspartate) receptors resulting in neuronal damage. The tissue-damaging process has been demonstrated in AIDS-related dementia complex, Alzheimer’s, stroke, epilepsy, and Huntington’s disease. Because Kynureninase is one of the key enzymes of QA production, its inhibitors may be useful for the treatment of neurological disorders. The recombinant Kynureninase has been shown to possess specificity for 3-hydroxykynurenine over kynurenine (4, 5).
- Lima, S. et al. (2007) Biochemistry 46:2735.
- Botting, N. P. (1995) Chem. Soc. Rev. 24:401.
- Stone, T. W. (2000) Trends in Pharm. Sci. 21:149.
- Walsh, H. et al. (2002) Eur. J. Chem. 269:2069.
- Toma, S. et al. (1997) FEBS Lett. 408:5.
Citation for Recombinant Human Kynureninase Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
HIV-1 Envelope Mimicry of Host Enzyme Kynureninase Does Not Disrupt Tryptophan Metabolism
Authors: Barton F Haynes
J. Immunol., 2016;0(0):.
Sample Types: Recombinant Protein
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