• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane

• Endotoxin Level

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

• Activity

  Measured by its ability to convert the substrate benzyloxycarbonyl-Gly-Pro-7-amido-4-methylcoumarin (Z-GP-AMC) to Z-Gly-Pro and 7-amino-4-methylcoumarin (AMC). The specific activity is >2000 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Mouse myeloma cell line, NS0-derived Leu26-Asp761 Accession # P97321

• Accession #

• N-terminal Sequence    
  Analysis

  Leu26 & Val31

• Predicted Molecular Mass

  85 kDa

• SDS-PAGE

  78-90 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM Tris, 1 M NaCl, 1 mg/mL BSA, pH 7.5

• Recombinant Mouse Fibroblast Activation Protein alpha /FAP (rmFAP) (Catalog # 8647-SE)

• Substrate: Z-Gly-Pro-AMC (Bachem, Catalog # I-1145), 10 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 rmFAP to 0.2 µg/mL in Assay Buffer.

2. Dilute Substrate to 100 µM in Assay Buffer.

3. Load 50 µL of 0.2 µg/mL of rmFAP into a plate, and start the reaction by adding 50 µL of 100 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate.

4. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.

5. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard 7-Amino, 4-Methyl Coumarin (Sigma, Catalog # A9891).

Per Well:

• rmFAP: 0.010 µg

• Substrate: 50 µM

Background: Fibroblast Activation Protein alpha/FAP

FAP (also known as seprase) is a 95 kDa Type II transmembrane serine protease that is structurally related to dipeptidyl peptidase IV (DPPIV/CD26) (1, 2). Within the extracellular domain, mouse FAP shares 90% and 97% amino acid (aa) sequence identity with human and rat FAP, respectively (3, 4). Alternative splicing of mouse FAP generates isoforms with a 33 aa or 5 aa deletion in the extracellular juxtamembrane region (3). FAP is expressed on reactive stromal fibroblasts in tumor tissue and wound healing and on synoviocytes in rheumatoid arthritis (1, 5-7). It exhibits dipeptidyl peptidase activity with substrate specificity similar to DPPIV, which is specific for N-terminal Xaa-Pro sequences (5, 8). FAP is also an endopeptidase that can degrade Gelatin, Collagens I and IV, Fibronectin, and Laminin (1, 5, 8) as well as several peptide hormones (  e.g. Neuropeptide Y, Brain Natriuretic Peptide, Substance P, Peptide YY, and Incretins) (9). The enzymatic activity is dependent on FAP association with DPPIV on the cell surface (5, 8, 10, 11). The matrix-dedgrading activity of FAP contributes to tumor cell migration and invasion (10-13). In addition, FAP can enhance tumor cell growth by limiting the development of anti-tumor immunity (14).

• References:

1. Zi, F. et al. (2015) Mol. Med. Rep. 11:3203.

2. Pineiro-Sanchez, M.L. et al. (1997) J. Biol. Chem. 272:7595.

3. Niedermeyer, J. et al. (1997) Int. J. Cancer 71:383.

4. Scanlan, M.J. et al. (1994) Proc. Natl. Acad. Sci. USA 91:5657.

5. Park, J.E. et al. (1999) J. Biol. Chem. 274:36505.

6. Rettig, W.J. et al. (1988) Proc. Natl. Acad. Sci. USA 85:3110.

7. Bauer, S. et al. (2006) Arthritis Res. 8:R171.

8. Aertgeerts, K. et al. (2005) J. Biol. Chem. 280:19441.

9. Keane, F.M. et al. (2011) FEBS J. 278:1316.

10. Ghersi, G. et al. (2006) Cancer Res. 66:4652.

11. Ghersi, G. et al. (2002) J. Biol. Chem. 277:29231.

12. Cheng, J.D. et al. (2005) Mol. Cancer Ther. 4:351.

13. Cheng, J.D. et al. (2002) Cancer Res. 62:4767.

14. Kraman, M. et al. (2010) Science 330:827.

• Entrez Gene IDs:

  2191 (Human); 14089 (Mouse)

• Alternate Names:

  170 kDa melanoma membrane-bound gelatinase; DKFZp686G13158; DPPIV; EC 3.4.21.-; FAP; FAPA; Fibroblast Activation Protein alpha; fibroblast activation protein, alpha; Integral membrane serine protease; Seprase; vibronectin