Recombinant Human Tripeptidyl-peptidase I/TPP1 Protein, CF

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

Recombinant Human Tripeptidyl-peptidase I/TPP1 Protein, CF Summary

• Details of Functionality: Measured by the cleavage of Ala-Ala-Phe-AMC. The specific activity is >850 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Tripeptidyl-Peptidase I/TPP1 protein
  Met1-Pro563, with a C-terminal 10-His tag
• Accession #: O14773
• N-terminal Sequence: Ser20
• Protein/Peptide Type: Recombinant Enzymes
• Gene: TPP1
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 61 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: 65-75 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 Tris and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • Activation Buffer: 50 mM NaOAc, 100 mM NaCl, pH 3.5
  • Assay Buffer: 50 mM NaOAc, 100 mM NaCl, pH 5.0
  • Recombinant Human Tripeptidyl‑Peptidase I/TPP1 (rhTPP1) (Catalog # 2237-SE)
  • Substrate: AAF-AMC (Bachem, Catalog # I-1415), 10 mM in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhTPP1 to 20 μg/mL in Activation Buffer.
  2. Incubate for 1 hour at 37 °C.
  3. Dilute rhTPP1 to 0.5 ng/μL in Assay Buffer.
  4. Dilute Substrate to 400 µM in Assay Buffer.
  5. Load into a black well plate 50 µL of the 0.5 μg/mL rhTPP1 and start the reaction by adding 50 µL of 400 µM substrate.
  6. Read at excitation and emssion wavelengths of 380 nm and 460 nM (top read), respectively, in kinetic mode for 5 minutes.
  7. 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 7-amino, 4-methyl coumarin (Sigma, Catalog # A-9891).

  Per Well:
  • rhTPP1: 0.025 µg
  • Substrate: 200 µM

Notes

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

Alternate Names for Recombinant Human Tripeptidyl-peptidase I/TPP1 Protein, CF

• Cell growth-inhibiting gene 1 protein
• ceroid-lipofuscinosis, neuronal 2, late infantile (Jansky-Bielschowsky disease)
• CLN2
• CLN2EC 3.4.14.9
• growth-inhibiting protein 1
• LINCL
• LPIC
• lysosomal pepstatin insensitive protease
• Lysosomal pepstatin-insensitive protease
• MGC21297
• TPP1
• TPP-1
• TPP-I
• Tripeptidyl aminopeptidase
• tripeptidyl peptidase I
• tripeptidyl-peptidase 1
• TripeptidylPeptidase I
• Tripeptidyl-Peptidase I

Background

Tripeptidyl-Peptidase I (TPP1), also known as tripeptidyl aminopeptidase, is a lysosomal peptidase which can hydrolyze tripeptides from the N-termini of oligopeptides and also possesses weak endopeptidase activity (1‑3). TPP1 is a serine peptidase with a Ser-Glu-Asp catalytic triad, making it a member of the sedolisin family (4). The TPP1 precursor undergoes autoactivation under conditions of acidic pH (4). TPP1 is expressed in many tissues, with elevated expression in tissues associated with peptide hormone production (5). Mutations in TPP1 have been shown to be a cause of classic late‑infantile neuronal ceroid lipofuscinosis (CLN2), a lysosomal storage disease (6).

1. Sleat, D.E. et al. (1997) Science 277:1802.
2. Ezaki, J. et al. (1999) J. Neurochem. 72:2573.
3. Tomkinson, B. (1999) Trends Biochem. Sci. 24:355.
4. Lin, L. et al. (2001) J. Biol. Chem. 276:2249.
5. Kida, E. et al. (2001) J. Neuropathol. Exp. Neurol. 60:280.
6. Sleat, D.E. et al. (1999) Am. J. Hum. Genet. 64:1511.         

Publications for Tripeptidyl-Peptidase I/TPP1 (2237-SE)(2)

Publications using 2237-SE

• N Sima, R Li, W Huang, M Xu, J Beers, J Zou, S Titus, EA Ottinger, JJ Marugan, X Xie, W Zheng Neural stem cells for disease modeling and evaluation of therapeutics for infantile (CLN1/PPT1) and late infantile (CLN2/TPP1) neuronal ceroid lipofuscinoses Orphanet J Rare Dis, 2018-04-10;13(1):54. 2018-04-10 [PMID: 29631617] (Bioassay, Human)
• Intravitreal implantation of TPP1-transduced stem cells delays retinal degeneration in canine CLN2 neuronal ceroid lipofuscinosis Exp Eye Res, 2016-09-13;0(0):. 2016-09-13 [PMID: 27637672] (Enzyme Assay, Human)

Bioinformatics

• Gene Symbol: TPP1
• Uniprot:
  • O14773()