MMP-14 Protein, Human (His-SUMO)

MMP-14 protein is an endopeptidase that degrades collagen and activates progelatinase A. MMP-14 Protein, Human (His-SUMO) is the recombinant human-derived MMP-14 protein, expressed by E. coli , with N-6*His, N-SUMO labeled tag.

MMP-14 Protein serves as a critical endopeptidase, targeting various components of the extracellular matrix, notably collagen, and playing a pivotal role in pericellular collagenolysis during development, contributing to the modeling of both skeletal and extraskeletal connective tissues. Additionally, MMP-14 activates progelatinase A, acting as a key player in the intricate regulation of the extracellular matrix. Beyond its matrix-degrading functions, MMP-14 is implicated in actin cytoskeleton reorganization by cleaving PTK7, highlighting its involvement in cellular processes beyond matrix remodeling. Furthermore, MMP-14 acts as a positive regulator of cell growth and migration through the activation of MMP15 and plays a crucial role in the formation of fibrovascular tissues in conjunction with pro-MMP2. Notably, MMP-14 cleaves ADGRB1, releasing vasculostatin-40, which exerts anti-angiogenic effects by inhibiting angiogenesis. This multifaceted functionality underscores MMP-14's significance in orchestrating diverse cellular processes and its potential as a therapeutic target in various physiological contexts.

Measured by its ability to cleave a fluorogenic peptide substrate Mca-KPLGL-Dpa-AR-NH2. The specific activity is 3373.72 pmol/min/µg, as measured under the described conditions. (Activation description: The proenzyme needs to be activated by Furin, Human (HY-P78747) for an activated form.)

Materials
Activation buffer: 50 mM Tris, 1 mM CaCl₂, 0.5% (w/v) Brij-35, pH 9.0
Assay buffer：50 mM Tris, 3 mM CaCl₂, 1μM ZnCl₂, pH 8.5
MMP-14 Protein, Human (His-SUMO) (HY-P700007)
Furin/PCSK3 Protein, Human (HEK293, His) (HY-P78747)
Substrate：MCA-Lys-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 (HY-P4931)
Standard: MOCAc-PLGL(Dpa)AR (HY-131498)

Procedure
1. Standard Curve Preparation: Dilute standard in assay buffer to concentrations of 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078, and 0 μmol/L. Add 100 μL of each dilution to a 96-well plate. Measure fluorescence in kinetic mode for 5 minutes at excitation and emission wavelengths of 320 nm and 405 nm, respectively. Plot the standard concentrations on the x-axis and the measured RFU values on the y-axis to generate a standard curve.
2. Activate 80 μg/mL Human MMP-14 with 1.72 μg/mL Human Furin in activation buffer.
3. Incubate at 37°C for 1.5 hours.
4. Dilute the activated Human MMP-14 to 1 ng/μL in detection buffer.
5. Dilute the substrate to 80 μM in detection buffer.
6. Experimental group: 50 μL of 1 ng/μL Human MMP-14 + 50 μL of 80 μM substrate.
Control group: 50 μL of detection buffer + 50 μL of 80 μM substrate.
7. Read in kinetic mode for 4 minutes at excitation wavelength 320 nm and emission wavelength 405 nm.
8. Calculate the specific activity:

Human

E. coli

P50281 (Y112-V582)

4323  [NCBI]

Full Length of Mature Protein

YAIQGLKWQHNEITFCIQNYTPKVGEYATYEAIRKAFRVWESATPLRFREVPYAYIREGHEKQADIMIFFAEGFHGDSTPFDGEGGFLAHAYFPGPNIGGDTHFDSAEPWTVRNEDLNGNDIFLVAVHELGHALGLEHSSDPSAIMAPFYQWMDTENFVLPDDDRRGIQQLYGGESGFPTKMPPQPRTTSRPSVPDKPKNPTYGPNICDGNFDTVAMLRGEMFVFKERWFWRVRNNQVMDGYPMPIGQFWRGLPASINTAYERKDGKFVFFKGDKHWVFDEASLEPGYPKHIKELGRGLPTDKIDAALFWMPNGKTYFFRGNKYYRFNEELRAVDSEYPKNIKVWEGIPESPRGSFMGSDEVFTYFYKGNKYWKFNNQKLKVEPGYPKSALRDWMGCPSGGRPDEGTEEETEVIIIEVDEEGGGAVSAAAVVLPVLLLLLVLAVGLAVFFFRRHGTPRRLLYCQRSLLDKV

Approximately 69.9 kDa.

Greater than 90% as determined by reducing SDS-PAGE.

Lyophilized powder.

Lyophilized from a 0.22 μm filtered solution of 10 mM Tris-HCl, 1 mM EDTA, 6% trehalose, pH 8.0.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O.

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.