Biotinidase/BTD Protein, Human (HEK293, His)

Biotinidase is our subject of interest, playing a key role in the catalytic release of biotin from biocytin, a by-product formed during biotin-dependent carboxylase degradation. This enzymatic process is essential to recycle biotin, an important cofactor, and ensure its participation in various carboxylation reactions. Biotinidase/BTD Protein, Human (HEK293, His) is the recombinant human-derived Biotinidase/BTD protein, expressed by HEK293 , with C-10*His labeled tag.

Biotinidase, encoded by the BTD gene, is an enzyme that plays a crucial role in catalyzing the release of biotin from biocytin, which is the product of the degradation of biotin-dependent carboxylases. This enzymatic activity is essential for recycling biotin, a water-soluble B-vitamin, from biocytin, allowing it to be utilized again by biotin-dependent carboxylases in various metabolic pathways. Biotinidase deficiency, a rare genetic disorder, can lead to the impaired recycling of biotin, resulting in a range of neurological and cutaneous symptoms. The catalytic function of biotinidase underscores its significance in maintaining biotin homeostasis and ensuring the continuous availability of biotin for various biochemical processes in the cell.

Measured by its ability to hydrolyze the substrate biotin 4-Nitrophenyl ester (BNP). The specific activity is 99.92 pmoL/min/μg, as measured under the described conditions.

Materials
Assay buffer: 50 mM Tris, 0.2 M NaCl, 0.1% Triton, pH 7.5
Biotinidase/BTD Protein, Human (HEK293, His) (HY-P72857)
Substrate: (+) -Biotin 4-Nitrophenyl ester (BNP)
Standard: 4-Nitrophenol

Procedure
1. Dilute 4-Nitrophenol (standard) in assay buffer to concentrations of 0, 125, 250, 500, 1000, 2000, 4000, 8000, 16000 pmol.
2. Add 100 μL of each concentration to the wells.
3. Measure the OD value at 405 nm using a microplate reader.
4. Plot the measured OD values on the x-axis and the concentration of the standard (pmol) on the y-axis to create the standard curve and obtain the standard curve equation.
5. Dilute rhBTD to 20 μg/mL in assay buffer.
6. Dilute BNP to 1 mM in DMSO.
7. Add 50 μL of 20 μg/mL Human BTD to the wells of a 96-well plate, followed by 50 μL of 1 mM BNP to initiate the reaction. For the blank wells, add 50 μL of assay buffer and 50 μL of 1 mM BNP.
8. Incubate at room temperature in the dark for 10 minutes.
9. Measure the OD value at 405 nm using a microplate reader.
10. Calculate enzyme activity:

Human

HEK293

C-10*His

P43251 (A42-D543)

686  [NCBI]

Full Length of Mature Protein

AHTGEESVADHHEAEYYVAAVYEHPSILSLNPLALISRQEALELMNQNLDIYEQQVMTAAQKDVQIIVFPEDGIHGFNFTRTSIYPFLDFMPSPQVVRWNPCLEPHRFNDTEVLQRLSCMAIRGDMFLVANLGTKEPCHSSDPRCPKDGRYQFNTNVVFSNNGTLVDRYRKHNLYFEAAFDVPLKVDLITFDTPFAGRFGIFTCFDILFFDPAIRVLRDYKVKHVVYPTAWMNQLPLLAAIEIQKAFAVAFGINVLAANVHHPVLGMTGSGIHTPLESFWYHDMENPKSHLIIAQVAKNPVGLIGAENATGETDPSHSKFLKILSGDPYCEKDAQEVHCDEATKWNVNAPPTFHSEMMYDNFTLVPVWGKEGYLHVCSNGLCCYLLYERPTLSKELYALGVFDGLHTVHGTYYIQVCALVRCGGLGFDTCGQEITEATGIFEFHLWGNFSTSYIFPLFLTSGMTLEVPDQLGWENDHYFLRKSRLSSGLVTAALYGRLYERD

75-90 kDa

• 
  ≥ 95%, as determined by reducing SDS-PAGE.

Lyophilized powder.

Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O. For long term storage it is recommended to add a carrier protein (0.1% BSA, 5% HSA, 10% FBS or 5% Trehalose).

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.