In vitro collagenase activity assay

1. Construct the expression vector (pET21a, Novagen) for His-tagged recombinant CiMMP2/9/13 (rCiMMP2/9/13)
2. Induce the rCiMMP2/9/13 expression in Rosseta 2 (DE3) (Novagen)
3. Sonicate Escherichia coli and extract rCiMMP2/9/13 in 100 mM Tris-HCl (pH 7.5)
4. Centrifuge the extract (9,000 rpm, 4°C, 15 min) and collect the precipitates
5. Suspend the precipitates in a lysis buffer and solubilize it by sonication
6. Purify the rCiMMP2/9/13 using Ni-NTA agarose (QIAGEN)
7. Dilute urea with PBS (pH 7.4) for approximately 20000-fold using Amicon Ultra-centrifugal filter (Merck)
8. Determine a concentration of rCiMMP2/9/13 using BCA Protein Assay kit (Thermo)
9. Mix 0.5 mg of rCiMMP2/9/13 with 5 mg of DQ collagen type I (Thermo, D12060) or type IV (Thermo, D12052) in a total volume of 200 ml reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl₂) in a presence of 1 mM MMP-2/MMP-9 inhibitor II (Calbiochem, 444249) or its solvent DMSO
10. Collagenase reaction should be done in triplicates or more.
11. Incubate at 20°C for 48 hr in dark
12. Transfer the samples to 96-well black plate (Corning, 3603) and measure the fluorescence using a FlexStation II (Molecular Devices) with the excitation wavelength of 485 nm, emission wavelength of 538 nm, and cut-off wavelength of 495 nm.

Solutions:

1. Lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 6 M urea, pH 8.0)
2. Reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl₂, pH 7.6)

Category