Recombinant Human MMP-16/MT3-MMP Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human MMP-16/MT3-MMP Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a fluorogenic peptide substrate Mca-KPLGL-Dpa-AR-NH₂ (Catalog # ES010). The specific activity is >250 pmol/min/µg, as measured under the described conditions.
• Source: E. coli-derived human MMP-16/MT3-MMP protein
  Ala32-Gly291 (Ile152Asn), with a C-terminal 10-His tag
• Accession #: P51512
• N-terminal Sequence: Ala32
• Structure / Form: Pro and catalytic domains
• Protein/Peptide Type: Recombinant Enzymes
• Gene: MMP16
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 31 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 29 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl, CaCl₂, ZnCl₂ and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • Activation Buffer: 50 mM Tris, 1 mM CaCl₂, 0.5% (v/v) Brij-35, pH 9.0
  • Assay Buffer: 50 mM Tris, 3 mM CaCl₂, 1 µM ZnCl₂, pH 8.5
  • Recombinant Human MMP‑16/MT3‑MMP (rhMMP-16) (Catalog # 1785-MP)
  • Recombinant Human Furin (rhFurin) (Catalog # 1503-SE)
  • Substrate: MCA-Lys-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH₂ (Catalog # ES010)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhMMP-16 to 80 µg/mL in Activation Buffer.
  2. Dilute rhFurin to 1.76 µg/mL in Activation Buffer.
  3. Mix equal volumes of 80 µg/mL of rhMMP-16 and 1.76 µg/mL of rhFurin together.
  4. Incubate at 37 °C for 1 hour to activate rhMMP-16.
  5. After incubation, dilute activated rhMMP-16 to 2 ng/µL in Assay Buffer.
  6. Dilute Substrate to 20 µM in Assay Buffer.
  7. In plate, load 50 µL of 2 ng/µL rhMMP-16, and start the reaction by adding 50 µL of 20 µM Substrate to wells. As a Substrate Blank, load 50 µL of Assay Buffer and 50 µL of 20 µM Substrate.
  8. Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes.
  9. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank
  **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

  Per Well:
  • rhMMP-16: 0.1 µg
  • Substrate: 10 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human MMP-16/MT3-MMP Protein, CF

• chromosome 8 open reading frame 57
• DKFZp761D112
• EC 3.4.24
• EC 3.4.24.-
• EC 3.4.24.80
• matrix metallopeptidase 16 (membrane-inserted)
• matrix metalloproteinase 16 (membrane-inserted)
• matrix metalloproteinase-16
• Membrane-type matrix metalloproteinase 3
• Membrane-type-3 matrix metalloproteinase
• MMP16
• MMP-16
• MMPX2
• MMP-X2
• MT3MMP
• MT3-MMP
• MT3-MMPC8orf57
• MT-MMP 3
• MT-MMP2
• MTMMP3
• MT-MMP3
• Putative transmembrane protein C8orf57

Background

Matrix metalloproteinases (MMPs) are a family of zinc and calcium dependent endopeptidases with the combined ability to degrade all the components of the extracellular matrix (ECM). MMP‑16 (MT3-MMP) is found in brain, lung, placenta, smooth muscle cells, and malignant tumor tissues including oral melanoma and renal carcinoma (1). MMP‑16 has been shown to activate proMMP-2 and degrade various ECM components including native collagens (2, 3). MMP‑16 has been proposed to possess the potential to directly enhance the growth and invasiveness of cells in vivo, two critical processes for development and carcinogenesis (4). Structurally, MMP‑16 consists of the following domains: a pro domain containing the furin cleavage site, a catalytic domain containing the zinc-binding site, a hinge region, a hemopexin-like domain, a transmembrane domain, and a cytoplamasic tail (1). The structure of the catalytic domain in complex with a hydroxamate inhibitor has been solved (5). The recombinant human MMP‑16PC consists of the pro and catalytic domains, which can be activated by treatment with furin.

1. Takino, T. et al. (1995) J. Biol. Chem. 270:23013.
2. Shofuda, K. et al. (1997) J. Biol. Chem. 272:9749.
3. Shimada, T. et al. (1999) Eur. J. Biochem. 262:907.
4. Kang, T. et al. (2000) FASEB J. 14:2559.
5. Lang, R. et al. (2004) J. Mol. Biol. 336:213.

Publications for MMP-16/MT3-MMP (1785-MP)(3)

Publications using 1785-MP

• H Yano, D Nishimiya, Y Kawaguchi, M Tamura, R Hashimoto Discovery of potent and specific inhibitors targeting the active site of MMP-9 from the engineered SPINK2 library PLoS ONE, 2020-12-29;15(12):e0244656. 2020-12-29 [PMID: 33373399] (Bioassay, Human)
• K Falkowski, E Bielecka, IB Thøgersen, O Boche?ska, K P?aza, M Kali?ska, L S?siadek, M Magoch, A P?cak, M Wi?niewska, N Gruba, M Wysocka, A Wojtysiak, M Brzezi?ska, K Sychowska, A Pejkovska, M Rehders, G Butler, CM Overall, K Brix, G Dubin, A Lesner, A Kozik, JJ Enghild, J Potempa, T Kantyka Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)-A CleavEx Based Analysis Int J Mol Sci, 2020-06-19;21(12):. 2020-06-19 [PMID: 32575583] (Bioassay, Human)
• Tsuchiya S, Simmer JP, Hu JC, Richardson AS, Yamakoshi F, Yamakoshi Y Astacin proteases cleave dentin sialophosphoprotein (Dspp) to generate dentin phosphoprotein (Dpp). J. Bone Miner. Res., 2011-01-01;26(0):220. 2011-01-01 [PMID: 20687161] (Bioassay, Human)

Bioinformatics

• Gene Symbol: MMP16
• Uniprot:
  • P51512()