Stromelysin-1/MMP-3 蛋白, 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 是一种金属蛋白酶，具有广泛的底物特异性，能够降解细胞外基质 (ECM) 的各种成分，例如纤连蛋白、层粘连蛋白、明胶（I、III、IV 和 V 型）、胶原蛋白（ III、IV、X 和 IX) 和软骨蛋白聚糖。这种酶在激活不同分子中发挥着关键作用，包括生长因子、纤溶酶原或其他基质金属蛋白酶（如 MMP9）。释放到 ECM 后，无活性的酶原通过纤溶酶级联信号通路进行激活。Stromelysin-1/MMP-3 也在细胞内发挥作用，正如在多巴胺能神经元中观察到的那样，在应激过程中，它被丝氨酸蛋白酶 HTRA2 激活，通过介导小胶质细胞激活和 α-突触核蛋白/SNCA 裂解，导致多巴胺神经元变性。此外，这种金属蛋白酶在免疫反应中发挥作用，并对多种病毒表现出抗病毒活性，包括水泡性口炎病毒、甲型流感病毒 (H1N1) 和人类疱疹病毒 1。从机制上讲，它在病毒感染时从细胞质转位到细胞核中调节 NF-kappa-B 活性。

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

• 
  Greater than 95% as determined by reducing SDS-PAGE.

溶液.

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.