Recombinant Human Biotinidase/BTD Protein, CF Summary
Details of Functionality |
Measured by its ability to hydrolyze the substrate biotin 4-Nitrophenyl ester (BNP). The specific activity is >190 pmol/min/μg, as measured under the described conditions. |
Source |
Chinese Hamster Ovary cell line, CHO-derived human Biotinidase/BTD protein Ala42-Asp543, with a C-terminal 6-His tag |
Accession # |
|
N-terminal Sequence |
Ala42 |
Protein/Peptide Type |
Recombinant Enzymes |
Gene |
BTD |
Purity |
>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane. |
Endotoxin Note |
<1.0 EU per 1 μg of the protein by the LAL method. |
Applications/Dilutions
Dilutions |
|
Theoretical MW |
58 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors. |
SDS-PAGE |
65-80 kDa, reducing conditions |
Packaging, Storage & Formulations
Storage |
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.- 6 months from date of receipt, -70 °C as supplied.
- 3 months, -70 °C under sterile conditions after opening.
|
Buffer |
Supplied as a 0.2 μm filtered solution in NaH2PO4, NaCl, EDTA and DTT. |
Purity |
>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane. |
Assay Procedure |
- Assay Buffer: 50 mM Tris, 0.2 M NaCl, 0.1% Triton, pH 7.5
- Recombinant Human Biotinidase/BTD (rhBTD) (Catalog # 7839-BT)
- (+)-Biotin 4-Nitrophenyl ester (BNP) (Sigma, Catalog # 861650), 50 mM stock in DMSO
- Dimethyl Sulfoxide (DMSO) (Sigma, Catalog # 34869)
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhBTD to 20 µg/mL in Assay Buffer.
- Dilute room temperature BNP to 1 mM in DMSO. Mix well.
- Load 50 µL of 20 µg/mL rhBTD in a clear strip well plate, and start the reaction by adding 50 µL of 1 mM BNP. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 1 mM BNP.
- Incubate sealed plate at room temperature for 10 minutes in the dark.
- Read at 405 nm (absorbance) in endpoint mode.
- Calculate specific activity:
Specific Activity (pmol/min/µg) = | Adjusted Abs* (OD) x Conversion Factor** (pmol/OD) | Incubation time (min) x amount of enzyme (µg) |
*Adjusted for Substrate Blank **Derived using calibration standard p-Nitrophenol (Sigma, Catalog # 241326). Per Well: |
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Biotinidase/BTD Protein, CF
Background
Biotinidase (BTD) is a member of the nitrilase superfamily, which consists of 12 families of nitrilases, amidases, carbamylases, and N‑acyltrasferases (1). It is a thiol hydrolase releasing biotin from biotinamide, biotin‑lysine, biotin‑peptide conjugates and biotin methylester. It is expressed in most mammalian tissues, with high activity being present in liver, kidney, serum, intestine, and adrenal glands (2). BTD with two other proteins, sodium-dependent multivitamin transporter, and holocarboxylase synthetase, play major roles in the homeostasis of biotin (3). BTD contributes to the homeostasis through the intestinal release of free biotin from digested biotin‑containing proteins and plasma transport and the recycling of biotin from breakdown products of biotinylated carboxylases. BTD deficiency can lead to a decrease in biotin bioavailability due to failure in releasing biotin from dietary proteins. It can be caused by gene mutations or from decreased secretion of BTD into the intestinal lumen (4). A recent study indicates that the BTD level in human plasma is a potential biomarker for the detection of breast cancer (5).
- Pace, H. C. and C. Brenner (2001) Genome. Biol. 2:reviews/0001.1.
- Chauhan, J. and K. Dakshinamurt (1986) J. Biol. Chem. 261:4286.
- Wolf, B. (2005) J Nur Biochem. 16:441.
- Zempleni, J. et al. (2008) Expert Rev Endocrinol Metab. 3:715.
- Kang, U. B. et al. (2010) BMC Cancer 10:114.
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