2/12/2016
Refolding and Purifying Recombinant MMP-12 Catalytic Domain
Mark O. Palmier
Dissolving the inclusion body pellet
1. Finely suspend the washed inclusion body pellet from a 750 ml fermentation in
17ml of 20mM Tris pH 4.9.
2. The conductivity should be no higher than 6 mS/cm before proceeding to step 3.
3. Add 12.6 g of urea and stir until completely dissolved. Continue stirring at room
temperature for 15 minutes.
4. The solution should fit perfectly (~35ml) into a 50 ml oakridge centrifuge tube.
5. Centrifuge the solution at 25000 x g for 45 minutes. The supernatant will be the
load solution for the next step.
Cation exchange purification of MMP-12 from inclusion body solution
Prepare 1 liter of chromatography buffers A and B.
Buffer A: 6M urea, 20 mM Tris-Acetate pH 4.5
Buffer B: BufferA adjusted to 1 M NaCl
1. Equilibrate a 40ml bed volume column of clean, ‘Fast Flow S-sepharose’ with
200 ml of Buffer A.
2. Pass the load solution from the the inclusion body step at a flow rate of 1 ml/min
at room temperature.
3. Wash the resin with 200 ml of Buffer A or until the baseline (A280) reaches a
steady, low absorbance (~0).
4. Gradient elute the resin from 0- 100% Buffer B over a period of 30 minutes using
a flow rate of 1.5 ml/min.
5. Check the fractions against the A280 elution trace by analyzing them with SDSPAGE.
6. Combine fractions containing the most concentrated and relatively pure MMP-12.
Remember that this is a preliminary purification step designed to remove gross
impurities, especially large polynucleic acids. Hence, this step will not
necessarily yield very high purity MMP-12.
Figure 1.
Elution profile
of a cation
exchange
purification of
inclusion
bodies.
1
2/12/2016
Mark O. Palmier
Refolding purified denatured MMP-12
Prepare 1. Refolding buffer: 6 M urea, 20 mM Tris–HCl, 5 mM CaCl2, and 100 mM NaCl,
pH 7.5.
2. Combine chosen fractions from the s-sepharose purification, dilute with refolding
buffer to a protein concentration of 0.1 mg/ml.
The resultant diluted protein is dialyzed in following steps:
1. Dialyze against refolding buffer containing 3, 1, and 0 M urea successively with
each dialysis performed at 4ºC for 4 h against 5 volumes of the dialysis buffer.
2. Dialyze against 20 mM Tris–HCl, 5 mM CaCl2, pH 7.3 at 4ºC for 4 hours.
3. Add 40 ml of clean, water-hydrated s-sepharose and rock for 10 minutes in the
orbital shaker set at 10ºC. (Note: The resin is simply water hydrated with no
buffer agent.)
4. Adjust the slurry to 100μM ZnCl2 and rock for 45 minutes.
5. Pour into an empty column and wash with 20 mM Tris–HCl, 5 mM CaCl2, 0.1
mM ZnCl2 pH 7.3 until the baseline is zero or flat after passing at least one resin
volume of the buffer. This step and the following steps are performed at room
temperature.
6. Elute the folded MMP-12 with a linear gradient of 0 – 1M NaCl over 30 minutes
using a 1.5 ml/min flow rate.
7. Collect 8 ml fractions and analyze suitable fractions by SDS-PAGE.
8. Determine [MMP-12] in the high purity fractions by the BioRad protein assay.
9. Check the activity of the renatured MMP-12 by kinetic analysis with FS-6.
10. Adjust the final pool of MMP-12 to 50% glycerol w/v, freeze aliquots and store at
-80ºC.
Figure 2.
Elution profile
of a cation
exchange
purification of
the refolded
MMP-12.
2
2/12/2016
Mark O. Palmier
This enzyme preparation should stay active for at least one year with no detectable loss in
catalytic activity when assayed with FS-6. The specific activity of wt MMP-12 CD with
FS-6 is 133800 M-1 s-1.
If the specificity constant (kcat/Km) is known for the MMP-12 mutant then a simple
progress curve measuring activity with an [FS-6] << Km will suffice to measure the active
site concentration. If a kcat/Km value is not known then a full active site titration must be
done with a suitable fast, tight binding inhibitor like galardin (GM6001).
Data: Lot0313082052_Intensity
Model: kcat_Km_v2
Weighting:
y
No weighting
1000000
900000
800000
Chi^2/DoF
= 4575766.65813
R^2
= 0.99978
RFU
700000
125nM
T205K H206D
MMP-12, 4M FS-6
600000
From previous AST the
-1 -1
kcat/Km = 118290 ± 298 M s
500000
400000
This activity measurement indicates an enzyme
concentration of 121nM
300000
0
50
100
150
200
250
300
350
time (s)
3
400
Fx
k
Et
B
921973 ±0
118290 ±0
1.2108E-7
330932.9
±1.054E-10
±0