Stromelysin-1/MMP-3 Protein, Human (HEK293, His)

The Stromelysin-1/MMP-3 protein is a multifunctional metalloprotease that degrades a variety of extracellular matrix components and activates molecules such as growth factors, plasminogen, and MMP9. It is released into the ECM and is activated through the plasmin cascade. Stromelysin-1/MMP-3 Protein, Human (HEK293, His) is the recombinant human-derived Stromelysin-1/MMP-3 protein, expressed by HEK293 , with C-6*His labeled tag.

Stromelysin-1/MMP-3, a metalloproteinase, exhibits a broad substrate specificity, capable of degrading various components of the extracellular matrix (ECM) such as fibronectin, laminin, gelatins (type I, III, IV, and V), collagens (III, IV, X, and IX), and cartilage proteoglycans. This enzyme plays a pivotal role in activating different molecules, including growth factors, plasminogen, or other matrix metalloproteinases like MMP9. Upon release into the ECM, the inactive pro-enzyme undergoes activation through the plasmin cascade signaling pathway. Stromelysin-1/MMP-3 also functions intracellularly, as observed in dopaminergic neurons where it becomes activated by the serine protease HTRA2 during stress, contributing to dopamine neuronal degeneration by mediating microglial activation and alpha-synuclein/SNCA cleavage. Additionally, this metalloproteinase plays a role in immune response and exhibits antiviral activity against various viruses, including vesicular stomatitis virus, influenza A virus (H1N1), and human herpes virus 1. Mechanistically, it translocates from the cytoplasm into the cell nucleus upon virus infection to modulate NF-kappa-B activities.

Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH2. The specific activity is >150 pmol/min/µg, as measured under the described conditions. (Activation description: The proenzyme needs to be activated by Chymotrypsin for an activated form.)

Materials
Assay buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35, pH 7.5 (TCNB)
Stromelysin-1/MMP-3 Protein, Human (HEK293, His) (HY-P70139)
Chymotrypsin: Dissolved in 1 mM HCl to 1 mg/mL
PhenylmethylsμLfonyl fluoride (PMSF) : Dissolved in 2-propanol to 0.2 M
Substrate: Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (HY-P2185A), dissolved in DMSO to 2 mM
Standard: MCA-Pro-Leu-OH

Procedure
1. Dilute MCA-Pro-Leu-OH in Detection Buffer to concentrations of 50, 25, 12.5, 6.25, 3.125, 1.5625, 0 μmol/L. Add 100 μL of each concentration to the wells to initiate the reaction. Read the fluorescence in kinetic mode for 5 minutes with excitation and emission wavelengths of 320 nm and 405 nm (top read) . Plot the standard concentrations on the x-axis and the measured RFU on the y-axis to create the standard curve and obtain the standard curve equation.
2. Dilute Human MMP-3 in assay buffer to 5 μg/mL and activate with Chymotrypsin to 20 μg/mL.
3. Incubate the reaction at 37°C for 30 minutes.
4. Stop the activation with 2 mM PMSF. Preheat the PMSF to 37°C before adding to the samples.
5. Dilute activated Human MMP-3 to 2.5 μg/mL in assay buffer.
6. Dilute the substrate to 20 μM in assay buffer.
7. Add 50 μL of 2.5 μg/mL Human MMP-3 to a black plate, and initiate the reaction by adding 50 μL of 20 μM substrate. For blank wells, add 50 μL of assay buffer and 50 μL of 20 μM substrate.
8. Read the fluorescence in kinetic mode for 5 minutes with excitation and emission wavelengths of 320 nm and 405 nm, respectively.
9. Calculate specific activity:

Human

HEK293

C-6*His

P08254 (Y18-C477)

4314  [NCBI]

Full Length of Mature Protein

YPLDGAARGEDTSMNLVQKYLENYYDLKKDVKQFVRRKDSGPVVKKIREMQKFLGLEVTGKLDSDTLEVMRKPRCGVPDVGHFRTFPGIPKWRKTHLTYRIVNYTPDLPKDAVDSAVEKALKVWEEVTPLTFSRLYEGEADIMISFAVREHGDFYPFDGPGNVLAHAYAPGPGINGDAHFDDDEQWTKDTTGTNLFLVAAHEIGHSLGLFHSANTEALMYPLYHSLTDLTRFRLSQDDINGIQSLYGPPPDSPETPLVPTEPVPPEPGTPANCDPALSFDAVSTLRGEILIFKDRHFWRKSLRKLEPELHLISSFWPSLPSGVDAAYEVTSKDLVFIFKGNQFWAIRGNEVRAGYPRGIHTLGFPPTVRKIDAAISDKEKNKTYFFVEDKYWRFDEKRNSMEPGFPKQIAEDFPGIDSKIDAVFEEFGFFYFFTGSSQLEFDPNAKKVTHTLKSNSWLNC

Approximately 60 kDa

• 
  ≥ 95%, as determined by reducing SDS-PAGE.

Solution.

Supplied as a 0.22 μm filtered solution of 20 mM Tris-HCl, 150 mM NaCl, 0.05% Brij35, 10% Glycerol, pH 7.5.
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.

N/A.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.

• [1]. /biology-dictionary/hct-116-cell.html  [Content Brief]