THOP1 Protein, Human (His)

The THOP1 protein metabolizes short neuropeptides, degrading them in the cytoplasm. It also degrades amyloid precursor protein, producing fragments associated with amyloid production. THOP1 Protein, Human (His) is the recombinant human-derived THOP1 protein, expressed by E. coli , with C-6*His labeled tag.

THOP1 Protein plays a crucial role in the metabolism of neuropeptides with a length of fewer than 20 amino acid residues, participating specifically in cytoplasmic peptide degradation. Its enzymatic activity extends to the degradation of the amyloid-beta precursor protein, generating fragments associated with amyloidogenesis. Additionally, THOP1 serves as a regulatory component in the cannabinoid signaling pathway, where it facilitates the degradation of hemopressin, an antagonist peptide targeting the cannabinoid receptor CNR1. The diverse functions of THOP1 underscore its involvement in intricate cellular processes, ranging from neuropeptide metabolism to the modulation of key signaling pathways.

Measured by its ability to cleave a fluorogenic peptide substrate Mca-PLGPK(Dnp)-OH that incubate at room temperature in kinetic mode for 5 minutes. The specific activity is 2815.75 pmol/min/µg.

Materials
Assay buffer: 25 mM Tris, 150 mM NaCl, pH 7.5
THOP1 Protein, Human (His) (HY-P71359)
Substrate: Mca-Pro-Leu-Gly-Pro-D-Lys(Dnp) (HY-P3792), dissolved in DMSO to 2 mM
Standard: MCA-Pro-Leu-OH

Procedure
1. Preparation of standard curve: Dilute MCA-Pro-Leu-OH to 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0 μmol/L respectively with assay buffer. Add 100 μL of each concentration standard to a 96-well plate. Read at wavelengths of 320 nm and 405 nm in kinetic mode (top read) for 5 minutes. Plot a standard curve with the standard concentration as the abscissa and the measured OD as the ordinate, and obtain the standard curve equation.
2. Dilute Human THOP1 to 500 ng/mL with assay buffer.
5. Dilute the substrate to 20 μM with assay buffer.
6. Add 50 μL of Human THOP1 to a 96-well plate, and add 50 μL of 20 μM substrate to initiate the reaction. For blank wells, add 50 μL of assay buffer and 50 μL of 20 μM substrate.
7. Read at wavelengths of 320 nm and 405 nm in kinetic mode (top read) for 5 minutes, respectively.
8. Calculate the specific activity:

Human

E. coli

C-6*His

P52888 (K2-C689)

7064  [NCBI]

Partial

KPPAACAGDMADAASPCSVVNDLRWDLSAQQIEERTRELIEQTKRVYDQVGTQEFEDVSYESTLKALADVEVTYTVQRNILDFPQHVSPSKDIRTASTEADKKLSEFDVEMSMREDVYQRIVWLQEKVQKDSLRPEAARYLERLIKLGRRNGLHLPRETQENIKRIKKKLSLLCIDFNKNLNEDTTFLPFTLQELGGLPEDFLNSLEKMEDGKLKVTLKYPHYFPLLKKCHVPETRRKVEEAFNCRCKEENCAILKELVTLRAQKSRLLGFHTHADYVLEMNMAKTSQTVATFLDELAQKLKPLGEQERAVILELKRAECERRGLPFDGRIRAWDMRYYMNQVEETRYCVDQNLLKEYFPVQVVTHGLLGIYQELLGLAFHHEEGASAWHEDVRLYTARDAASGEVVGKFYLDLYPREGKYGHAACFGLQPGCLRQDGSRQIAIAAMVANFTKPTADAPSLLQHDEVETYFHEFGHVMHQLCSQAEFAMFSGTHVERDFVEAPSQMLENWVWEQEPLLRMSRHYRTGSAVPRELLEKLIESRQANTGLFNLRQIVLAKVDQALHTQTDADPAEEYARLCQEILGVPATPGTNMPATFGHLAGGYDAQYYGYLWSEVYSMDMFHTRFKQEGVLNSKVGMDYRSCILRPGGSEDASAMLRRFLGRDPKQDAFLLSKGLQVGGCEPEPQVC

80-85 kDa

• 
  Greater than 95% as determined by reducing SDS-PAGE.

Solution.

Supplied as a 0.22 μm filtered solution of 20 mM Tris-HCl, 500 mM NaCl, 50% Glycerol, pH 7.4.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<1 EU/μg, determined by LAL method.

N/A.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.