• Purity

  >90%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to cleave a fluorogenic peptide substrate Mca-KPLGL-Dpa-AR-NH    ₂ (Catalog # ). The specific activity is >50 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Phe29-Pro462 (Tyr357His) with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Phe29, Ala63, Asp140

• Predicted Molecular Mass

  48 kDa

• SDS-PAGE

  55-76 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM NaOAc, 1.0 M NaCl, 0.05% Brij-35 (w/v), pH 3.0

• Recombinant Mouse BACE-2 (rmBACE-2) (Catalog # 2977-AS)

• Substrate: MCA-Lys-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH₂ (Catalog # )

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rmBACE-2 to 2 ng/μL in Assay Buffer.

2. Dilute Substrate to 20 μM in Assay Buffer.

3. Load 50 μL of the 2 ng/μL rmBACE-2 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:

     *Adjusted for Substrate Blank
     **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

Per Well:

• rmBACE-2: 0.1 μg

• Substrate: 10 μM

Background: BACE-2

BACE-2 is an aspartic protease that shares sequence similarity with BACE-1, the major beta ?secretase for generation of A beta peptide in neurons (1). However, BACE-2 differs from BACE-1 in several aspects including pro-enzyme activation, substrate binding sites, transcriptional regulation, and function in A beta peptide production (2?4). The domain structure of mouse BACE-2 consists of a signal peptide (aa 1?19), pro (aa 20?62) and a mature chain (aa 63?514) that consists of a transmembrane domain and a cytoplasmic tail. The C?terminal region (aa 463?514) was replaced by a His tag in the Recombinant Mouse BACE-2 that also contained a His residue at the position of Tyr357. The purified rmBACE-2 started at Ala63, the same residue shown at the N-terminus of the human active BACE-2 (2).

• References:

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. (2005) FASEB J. 19:739.

• Long Name:

  beta-site Ayloid Precursor Protein Cleaving Enzyme 2

• Entrez Gene IDs:

  25825 (Human); 56175 (Mouse); 288227 (Rat)

• Alternate Names:

  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