Recombinant Human BACE-2 Protein, CF

Recombinant Human BACE‑2 (Catalog # 4097-ASB) is measured by its ability to cleave a fluorogenic peptide substrate Mca-KPLGL-Dpa-AR-NH2 (Catalog # ES010).

2 μg/lane of Recombinant Human BACE‑2 was resolved with SDS-PAGE underreducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Bluestaining, showing a band at approximately 55 kDa under R conditions.

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

Recombinant Human BACE-2 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a fluorogenic peptide substrate Mca-KPLGL-Dpa-AR-NH₂ (Catalog # ES010). The specific activity is >70 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human BACE-2 protein
  Phe29-Pro466, with a C-terminal 10-His tag
• Accession #: Q9Y5Z0-1
• N-terminal Sequence: Phe29 & Ala63
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 49 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: 48-60 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 Tris and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM Sodium Acetate, 1 M NaCl, 0.05% Brij-35, pH 3.0
  • Recombinant Human BACE-2 (rhBACE-2) (Catalog # 4097-ASB)
  • Substrate: MCA-Lys-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH₂ (Catalog # ES010)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhBACE-2 to 2 ng/µL in Assay Buffer.
  2. Dilute Substrate to 50 µM in Assay Buffer.
  3. Load 50 µL of 2 ng/µL rhBACE-2 in a plate, and start the reaction by adding 50 µL of 50 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 50 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:
  • rhBACE-2: 0.1 µg
  • Substrate: 25 µM

Notes

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

Alternate Names for Recombinant Human BACE-2 Protein, CF

• AEPLC
• Asp 1
• ASP1
• ASP21
• Aspartic-like protease 56 kDa
• Aspartyl protease 1
• BACE2
• BACE-2
• beta secretase 2
• beta-secretase 2
• beta-site amyloid beta A4 precursor protein-cleaving enzyme 2
• Beta-site amyloid precursor protein cleaving enzyme 2
• Beta-site APP cleaving enzyme 2
• beta-site APP-cleaving enzyme 2
• beta-site APP-cleaving enzyme 2, EC 3.4.2310ALP5656 kDa aspartic-like protease
• CEAP1
• Down region aspartic protease
• Down syndrome region aspartic protease
• DRAPBAE2
• EC 3.4.23
• EC 3.4.23.45
• memapsin-1
• Membrane-associated aspartic protease 1
• theta-secretase
• transmembrane aspartic proteinase Asp1

Background

BACE-2 (Beta secretase 2) is an aspartic protease that shares 48% sequence identity with BACE-1 in the extracellular catalytic domains. BACE-1 is the putative beta secretase for the generation of the A beta peptide in neurons (1). BACE-2 differs from BACE-1 in several aspects, including proenzyme activation, substrate preference, transcriptional regulation, and expression pattern (2, 3). Unlike BACE-1, BACE-2 activity does not contribute to Alzheimer's disease pathogenesis (4) but has been shown to play a key role in insulin receptor trafficking in the pancreas where it is expressed in beta -cells (5,6). BACE-2 affects glucose tolerance and was suggested as a promising target for improving beta -cell function in diabetes (7). Recombinant human BACE-2 was expressed without its C terminal transmembrane and cytosolic domains, resulting in its secretion from NS0 cells.

1. Cai, H. et al. (2001) Nature Neurosci. 4:233.
2. Hussain, I. et al. (2001) J. Biol. Chem. 276:23322.
3. Ostermann, N. et al. (2006) J. Mol. Biol. 355:249.
4. Sun, X. et al. (2006) FASEB J. 19:739.
5. Esterhazy. D. et al. (2011) Cell Metab. 14:365.
6. Casas, S. et al. (2010). Am. J. Physiol. Endocrinol. Metab. 299:E1087.
7. Alcarraz-Vizan, G. et al. (2017). Cell Mol. Life Sci. 74:2827.

Bioinformatics

• Uniprot:
  • Q9Y5Z0-1()