Recombinant Human Kynureninase Protein, CF

Catalog #: 4887-KH
2 Citations Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
4887-KH-010

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Human Kynureninase Protein, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by silver stain

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to oxidize 3-hydroxy kynurenine. The specific activity is > 75 pmol/min/µg, as measured under the described conditions.

Source

Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Kynureninase protein
Met1-Asn465, with a C-terminal 10-His tag

Accession #

NP_003928

N-terminal Sequence
Analysis

Met1

Predicted Molecular Mass

54 kDa

SDS-PAGE

51 kDa, reducing conditions

Product Datasheets

4887-KH

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

4887-KH

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. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials

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 V_max* (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).

Per Well:

rhKYNU: 0.1 µg
Substrate: 100 µM

Reconstitution Calculator

Background: Kynureninase

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).

References

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.

Entrez Gene IDs

8942 (Human); 70789 (Mouse); 116682 (Rat)

Alternate Names

EC 3.7.1.3; KYNU; kynureninase (L-kynurenine hydrolase); Kynureninase; L-kynurenine hydrolase

Related Research Areas

Citations 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.

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