Recombinant Human THOP1 Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human THOP1 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a fluorogenic peptide substrate, (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Pro-D-Lys(2,4-dinitrophenyl)-OH or Mca-PLGPK(Dnp)-OH. The specific activity is >250 pmol/min/µg, as measured under the described conditions.
• Source: E. coli-derived human Thimet Oligopeptidase/THOP1 protein
  Lys2-Cys689, with an N-terminal Met and 6-His tag
• Accession #: NP_003240
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: THOP1
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 80 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: 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, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in MES, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 25 mM Tris, 150 mM NaCl, pH 7.5
  • Recombinant Human Thimet Oligopeptidase/THOP1 (rhTHOP1) (Catalog # 3439-ZN)
  • Substrate: MCA-Pro-Leu-Gly-Pro-D-Lys-(DNP)-OH (Bachem, Catalog # M-2270); 2 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: Spectramax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhTHOP1 to 0.5 ng/µL in Assay Buffer.
  2. Dilute Substrate to 20 µM in Assay Buffer.
  3. Load 50 µL of the 0.5 ng/µL rhTHOP1 in a plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 20 µM Substrate.
  4. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  5. 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 MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

  Per Well:
  • rhTHOP1: 0.025 µg
  • Substrate: 10 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human THOP1 Protein, CF

• EC 3.4.24
• EC 3.4.24.15
• Endopeptidase 24.15
• EP24.15
• MEPD_HUMAN
• MP78
• thimet oligopeptidase 1
• thimet oligopeptidase
• THOP1
• TOP

Background

Thimet Oligopeptidase/THOP1, also known as endopeptidase EC 3.4.24.15 (EP24.15), is a zinc peptidase of the M3 family that also includes neurolysin/EC 3.4.24.16 and mitochondrial intermediate peptidase (1). Widely expressed by mammalian tissues and reported to present in different subcellular locations, THOP1 is primarily a cytoplasmic enzyme. It is capable of hydrolyzing a number of bioactive peptides and peptides released by the proteosome, limiting antigenic presentation by MHC class I molecules (1-3). THOP1 also interacts with angiotensin II type I receptor and bradykinin B2 receptor (4). The optimal activity of the purified THOP1 may or may not require the presence of a reducing agent, depending upon the source of the enzyme and the purification method used.

1. Barrett, A.J. and J.-M. Chen (2004) in Handbook of Proteolytic Enzymes. Barrett, A.J. et al. eds. p. 352, Elsevier Academic Press, San Diego.
2. Ray, K. et al. (2004) J. Biol. Chem. 279:20480.
3. Saric, T. et al. (2004) J. Biol. Chem. 279:46723.
4. Shivakumar, B.R. et al. (2005) Cell Biochem. Funct. 23:195.

Publications for Thimet Oligopeptidase/THOP1 (3439-ZN)(1)

Publication using 3439-ZN

• Yanaika S. Hok‐A‐Hin, Katharina Bolsewig, Daimy N. Ruiters, Alberto Lleó, Daniel Alcolea, Afina W. Lemstra et al. Thimet oligopeptidase as a potential CSF biomarker for Alzheimer's disease: A cross‐platform validation study Alzheimer's & Dementia: Diagnosis, Assessment & Disease Monitoring 2023-07-26 [PMID: 37502019] (Simple Plex Automated Immunoassay, Human)

Bioinformatics

• Gene Symbol: THOP1
• Uniprot:
  • NP_003240()