• Purity

  >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

• 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 >1800 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Spodoptera frugiperda,     Sf 21 (baculovirus)-derived Leu26-Asp760, with an N-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  His

• Predicted Molecular Mass

  86 kDa

• SDS-PAGE

  85 kDa, reducing conditions

Assay Procedure

Materials

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

• Recombinant Human Fibroblast Activation Protein alpha /FAP (rhFAP) (Catalog # 3715-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 rhFAP 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 rhFAP 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 # A-9891).

Per Well:

• rhFAP: 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 (ECD), human FAP shares 90% amino acid (aa) sequence identity with mouse and rat FAP (3, 4). Alternative splicing of human FAP generates a truncated isoform that consists of the C-terminal 239 residues of the ECD. A soluble and enzymatically active form of FAP known as antiplasmin-cleaving enzyme (APCE) circulates in human plasma (4). FAP is expressed on reactive stromal fibroblasts in tumor tissue and wound healing and on synoviocytes in rheumatoid arthritis (1, 6-8). It exhibits dipeptidyl peptidase activity with substrate specificity similar to DPPIV, which is specific for N-terminal Xaa-Pro sequences (6, 9). FAP is also an endopeptidase that can degrade Gelatin, Collagens I and IV, Fibronectin, and Laminin (1, 6, 9) as well as several peptide hormones (  e.g. Neuropeptide Y, Brain Natriuretic Peptide, Substance P, Peptide YY, and Incretins) (10). The enzymatic activity is dependent on FAP association with DPPIV on the cell surface (6, 9, 11, 12). The matrix-dedgrading activity of FAP contributes to tumor cell migration and invasion (11-14). In addition, FAP can enhance tumor cell growth by limiting the development of anti-tumor immunity (15).

• 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