• Purity

  >80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie? Blue stain at 5 μg per lane

• Activity

  Measured by its ability to cleave a fluorogenic peptide substrate Mca-PLAQAV-Dpa-RSSSR-NH    ₂ (Catalog # ). The specific activity is >150 pmol/min/μg, as measured under the described condition. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Ala39-Ser531 (Arg125Pro) & (Ala182Thr), with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Ala39

• Predicted Molecular Mass

  47 kDa

• SDS-PAGE

  56-65 kDa, reducing conditions

Assay Procedure

Materials

• Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB) + 100 μM ZnCl₂

• Assay Buffer: 50 mM Tris, 10 mM CaCl₂, pH 7.5

• Recombinant Human MMP-17 (rhMMP-17) (Catalog # 7796-MP)

• p-aminophenylmercuric acetate (APMA) (Sigma, Catalog # A-9563), 100 mM stock in DMSO

• Substrate: MCA-PLAQAV-Dpa-RSSSR-NH₂ (Catalog # ), 2 mM stock in DMSO

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

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

1. Activate rhMMP-17 at 100 μg/mL with 1 mM APMA in Activation Buffer.

2. Incubate at room temperature for 2 hours.

3. Dilute activated rhMMP-17 to 2 ng/μL in Assay Buffer.

4. Dilute Substrate to 20 μM in Assay Buffer.

5. Load 50 μL of the 2 ng/μL rhMMP-17 into 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 without any rhMMP?17.

6. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.

7. Calculate specific activity:

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

Per Well:

• rhMMP-17: 0.1 μg

• Substrate: 10 μM

Background: MMP-17

Matrix metalloproteinase 17 (MMP?17) is a zinc metalloproteinase that is an integral membrane protein (1). It belongs to the membrane?type MMP (MT?MMP) subfamily, and is also known as MT4?MMP. It is associated with the cell membrane by a glycosylphosphatidylinositol (GPI) anchor (1). It has the potential to function as a pro?tumor necrosis factor?a  (TNF?a) convertase (2). Unlike MMP?14 (MT1?MMP), it is a poor activator of pro?MMP?2. MMP?17 is relatively poor at digesting components of the extracellular matrix, but does cleave fibrinogen and fibrin (2). MMP?17 is known to activate the aggrecanase ADAMTS?4 through proteolysis (3). MMP?17 is expressed in many tissues with the highest levels seen in the brain, colon, ovary, testis, and kidney papilla (4). It has been detected in several human cancers, including gliomas, prostate carcinomas, and breast carcinomas (5). Recombinant Human MMP?17 was expressed without its GPI anchoring sequence, resulting in its secretion as a soluble protein.

• References:

1. Itoh, Y. et al. (1999) J. Biol. Chem. 274:34260.

2. English, W. R. et al. (2000) J. Biol. Chem. 275:14046.

3. Gao, G. et al. (2004) J. Biol. Chem. 279:10042.

4. Srichai, M. B. et al. (2011) PLoS ONE 6:e17099.

5. Sohail, A. et al. (2008) Cancer Metastasis Rev.  27:289.

• Long Name:

  Matrix Metalloproteinase 17

• Entrez Gene IDs:

  4326 (Human); 23948 (Mouse); 288626 (Rat)

• Alternate Names:

  matrix metallopeptidase 17 (membrane-inserted); matrix metalloproteinase 17 (membrane-inserted); matrix metalloproteinase-17; membrane-type matrix metalloproteinase 4; membrane-type-4 matrix metalloproteinase; MMP17; MMP-17; MT4MMP; MT4-MMP; MT-MMP 4; MTMMP4