Animal-Free MMP-2 Protein, Human (His)

MMP-2 protein is a multifunctional metalloproteinase that actively participates in physiological processes such as vascular remodeling, angiogenesis, tissue repair, tumor invasion, inflammation, and atherosclerotic plaque rupture. In addition to degrading extracellular matrix proteins, it also acts on non-matrix proteins to promote vasoconstriction. Animal-Free MMP-2 Protein, Human (His) is the recombinant human-derived animal-FreeMMP-2 protein, expressed by E. coli , with C-His labeled tag. This product is for cell culture use only.

The MMP-2 protein, a ubiquitinous metalloproteinase, actively participates in a spectrum of physiological processes, including vasculature remodeling, angiogenesis, tissue repair, tumor invasion, inflammation, and atherosclerotic plaque rupture. Beyond its role in degrading extracellular matrix proteins, this protein demonstrates versatility by acting on non-matrix proteins, such as big endothelial 1 and beta-type CGRP, thereby promoting vasoconstriction. Additionally, it cleaves KISS at a Gly-|-Leu bond and appears to play a role in myocardial cell death pathways. By regulating the activity of GSK3beta and cleaving GSK3beta in vitro, it contributes to myocardial oxidative stress. In association with MMP14, MMP-2 is involved in the formation of fibrovascular tissues. Notably, the C-terminal non-catalytic fragment of MMP-2, known as PEX, possesses anti-angiogenic and anti-tumor properties, inhibiting cell migration and adhesion to FGF2 and vitronectin. Furthermore, it serves as a ligand for integrin alpha-v/beta3 on the surface of blood vessels.

Measured by its ability to cleave 20µM fluorogenic peptide substrate Mca-KPLGL-Dpa-AR-NH2 that incubate at room temperature in kinetic mode for 5 minutes .The specific activity is 855.17pmol/min/µg. (Activation description: The proenzyme needs to be activated by APMA (HY-148905) for an activated form.)

Materials
Assay buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
Animal-Free MMP-2 Protein, Human (His)（HY-P700139AF）
4-Aminophenylmercuric Acetate (APMA, HY-148905): Dissolved in DMSO to 100 mM
Substrate: MCA-Lys-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH₂(HY-131498)
Standard: MCA-Pro-Leu-OH

Procedure
1. Standard Curve: Dilute 7-amino, 4-Methyl Coumarin in assay buffer to concentrations of 0, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100 μmol/L. Add 100 μL of each concentration to the wells of a black microplate. Read the fluorescence in kinetic mode for 5 minutes with excitation and emission wavelengths of 320 nm and 405 nm (top read). Plot the measured RFU on the y-axis and the concentration of the standard (μmol/L) on the x-axis to create the standard curve and obtain the standard curve equation.
2. Dilute MMP-2 to 100 μg/mL in assay buffer containing 1 mM APMA.
3. Activation: Incubate at 37°C for 3 hours.
4. Dilute the activated MMP-2 protein to 2 ng/μL in assay buffer.
5. Dilute the substrate to 20 μM in assay buffer.
6. Experimental group: Mix 50 μL of 2 ng/μL MMP-2 protein with 50 μL of 20 μM substrate.
Control group: Mix 50 μL of 20 μM substrate with 50 μL of assay buffer.
7. Read the fluorescence in kinetic mode for 5 minutes with excitation and emission wavelengths of 320 nm and 405 nm, respectively.
8. Calculate specific activity:

Human

E. coli

C-6*His

P08253 (Y110-C660)

4313  [NCBI]

Full Length of Mature Protein

YNFFPRKPKWDKNQITYRIIGYTPDLDPETVDDAFARAFQVWSDVTPLRFSRIHDGEADIMINFGRWEHGDGYPFDGKDGLLAHAFAPGTGVGGDSHFDDDELWTLGEGQVVRVKYGNADGEYCKFPFLFNGKEYNSCTDTGRSDGFLWCSTTYNFEKDGKYGFCPHEALFTMGGNAEGQPCKFPFRFQGTSYDSCTTEGRTDGYRWCGTTEDYDRDKKYGFCPETAMSTVGGNSEGAPCVFPFTFLGNKYESCTSAGRSDGKMWCATTANYDDDRKWGFCPDQGYSLFLVAAHEFGHAMGLEHSQDPGALMAPIYTYTKNFRLSQDDIKGIQELYGASPDIDLGTGPTPTLGPVTPEICKQDIVFDGIAQIRGEIFFFKDRFIWRTVTPRDKPMGPLLVATFWPELPEKIDAVYEAPQEEKAVFFAGNEYWIYSASTLERGYPKPLTSLGLPPDVQRVDAAFNWSKNKKTYIFAGDKFWRYNEVKKKMDPGFPKLIADAWNAIPDNLDAVVDLQGGGHSYFFKGAYYLKLENQSLKSVKFGSIKSDWLGC

Approximately 63.00 kDa

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

Lyophilized powder.

Lyophilized from a 0.22 μm filtered solution of PBS, pH 8.0, trehalose.

<0.1 EU per 1 μg of the protein by the LAL method.

It is recommended to reconstitute to a concentration of 100-200 μg/mL in ddH₂O. For long term storage it is recommended to add a carrier protein (0.1% BSA, 5% HSA, 10% FBS or 5% Trehalose).

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.