MMP-7/PUMP1 His Tag Protein, Human

MMP-7 (Matrix Metalloproteinase-7), also known as Matrilysin or PUMP-1, is the smallest secreted zinc-dependent endopeptidase in the matrix metalloproteinase family, with a molecular weight of approximately 28 kDa and specific expression in epithelial cells. It has an extremely streamlined structure comprising only a signal peptide, pro-domain, and catalytic domain, lacking the hinge region and hemopexin-like domain commonly found in other MMPs. The HEXXHXXGXXH motif in its catalytic domain exerts proteolytic activity by chelating zinc ions, enabling it to degrade basement membrane components such as laminin, fibronectin, and type IV collagen, as well as activate inflammatory factors like pro-TNF-α and cleave E-cadherin to promote epithelial-mesenchymal transition. Under physiological conditions, it participates in tissue remodeling, wound healing, and gland development, whereas under pathological conditions, MMP-7 is highly expressed in various malignancies including gastric, colorectal, pancreatic, and renal cell carcinomas, closely correlating with poor prognosis through promoting tumor invasion, angiogenesis, and lymph node metastasis. Plasma MMP-7 level detection has demonstrated favorable diagnostic value (AUC 0.80-0.91) and independent prognostic predictive capability, and can assess pathological response to neoadjuvant therapy in pancreatic cancer patients, making it a highly promising tumor biomarker and therapeutic target.

Assay protocol

Principle: Measured by its ability to cleave a peptide substrate, Mca-PLGL-Dpa-AR-NH2.

Materials

1.Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij 35, pH 7.5 (TCNB)

2.MMP-7/PUMP1 His Tag Protein, Human (active enzyme)

3.p-aminophenylmercuric acetate (APMA) (Aladdin, Catalog # P649286)

4.Fluorogenic Peptide Substrate: MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 (R&D, ES001)

5.Calibration standard: MCA-Pro-Leu-OH (Shyuanye, T77046)

6.96 ELISA Removable Plate, Black, High binding (GENEVER, Catalog # GMO2-96H)

7.Plate Reader (PerkinElmer, excitation 320 nm, and emission 405 nm)

Produce

1.Activate MMP-7 at 100 μg/mL with 1 mM APMA in Assay Buffer. Incubate at 37 ℃ for 4 hours.

2.Dilute activated MMP-7 to 0.4, 0.2 and 0.1 μg/mL in Assay Buffer.

3.Dilute Substrate to 20 μM in Assay Buffer.

4.Load 50 μL of the dilute MMP-7 Protein (From the step 2) into a black well plate and start the reaction by adding 50 μL of 20 μM Substrate. Include a Substrate with Assay Buffer as Blank.

5.Read at excitation and emission wavelengths of 320 nm and 405 nm, kinetic mode, 60s/cycle, 20 cycles.

6.Calculate specific activity.

• Standard Curve

1. Dilute Calibration standard to 10 μM in Assay Buffer and prepare serial dilutions.

2. Add 100 ul of each serially diluted standard and blank (Assay buffer) into appropriate wells of a 96-well, the standard curve has a range of 1000, 500, 250, 125, 62.5, 31.25, 15.625 pmol per well.

3. Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively in endpoint mode.

4. Linear Regression of MCA-Pro-Leu-OH (pmol)(y) – RFU-Blank(x).