PURIFICATION OF PROCATHEPSIN D AND CATHEPSIN D USING
PEPSTATIN-AGAROSE
(see Conner, Biochem. J. 263: 601-604, (1989) and Conner, J.
Biol. Chem. 267: 21738-21745, (1992))
Cultured Cells
Baculovirus media
Human Placental Cathepsin D
How to make the pepstatin-agarose
GENERAL COMMENTS:
Starting material can be tissue extracts, culture media, cultured cells.
The most common problem in pepstatinyl agarose purification of cathepsin D arises from using too much pepstatinyl agarose and from eluting at high flow rates. This seems to dilute the enzyme and increase losses by absorption to surfaces. Make your own pepstatin-agrose. Calculate the expected amount of cathepsin D and use an appropriate amount of pepstatinyl agarose to bind all the enzyme. We typically use columns of 0.1 -0.5 ml pepstatinyl agarose. For example, the pepstatinyl agarose made by the procedure of Huang et al. (see below) should be at least 0.1-0.5 µmoles of pepstatin / ml based on 2-10% substitution of the available aminohexyl groups. Thus 1 ml of gel should bind at least 4 mg of cathepsin D.
Unreacted aminohexyl groups can bind nonspecifically through electrostatic or hydrophobic interactions - for this reason we run the column in high ionic strength, and if we anticipate really low amounts of enzyme (radiolabeled cell cultures) we sometimes include nonionic detergent.
Another frequent problem encountered in pepstatinyl agarose is slow release of pepstatin from the support during storage and subsequent loss of aspartic proteinase activity by soluble pepstatin. This is easily avoided by washing the beads in a large volume of binding buffer and then elution buffer. CNBr activated resins seem less stable than NHS-DCC activated resins.
We never batch bind samples unless they are small concentrated
samples as we have had poor success with this method. We do run
up to a liter of media or refolding mixtures (Conner and Richo
Biochemistry 31: 1142-1147 (1992)) through columns with bed
volumes as small of 0.2 ml. Slow and tedious but efficient.
For Cultured Cell Extracts
-
all procedures at 40 (at higher temps the procathepsin
D will activate)
60 mm dishes, washed with PBS, are harvested in 0.25 ml/ dish
(the high concentration is important for efficient binding) using
:
0.2% Triton X-100,
5 mM Tris-HCl pH 8.3
5 mM EDTA (neutralized)
add PMSF , leupeptin,
add BSA to 0.4 mg final, NaCl to 1 M final and Na formate pH3.5
to 0.1 M final
then add 20uL of 50% slurry Pepstatin agarose (ie 10 uL of beads)
incubate with rotation for 3 h
wash beads in:
0.1% triton X-100
0.125 M Na fromate pH3.5
0.35 M NaCl
elute in gel sample buffer or elute the column with elution buffer of your choice above pH 7.0 - we have successfully used phosphate and Tris between pHs of 7 and 8.5 with and without detergent or NaCl.
It is essential that
the column be eluted as slowly as possible-to prevent dilution of
the enzyme.
all at 40
Centrifuge media at low speed to remove cells (5-10 min at 1000-5000 xg)
Make the cleared media pH 3.5 - we do this by adding 0.1 vol of :
1M Na Formate pH3.5
1% Brij 35
2 M NaCl
(if you want to purify procathepsin D, the acidified sample should NEVER be allowed to go over 40)
After 1h at 40, centrifuge at high speed to remove virus and acid-precipitated proteins (at least 70,000 xg for 1 h) - this is an important purification step as virus represents a majority of the protein in serum free media.
apply to pepstatinyl -agarose (flow rate has not appeared to be an important factor, in fact a faster flow rate seems to improve purification as other proteins may continue to precipitate while the sample is being loaded).
wash the column with
0.1 M Na Formate pH 3.5
0.05% Brij 35
0.5 M Na Cl
then wash with
0.01 M Na Formate pH 3.5
0.05% Brij 35
0.5 M Na Cl
This latter wash drops the concentration of formate making pH
change during elution easier
elute the column with elution buffer of your choice above pH 7.0 - we have successfully used phosphate and Tris between pHs of 7 and 8.5 with and without detergent or NaCl.
It is essential that the column be eluted as slowly as possible-to prevent dilution of the enzyme.
Activation and Assay
To fully express activity incubate the purified enzyme at pH 3.5
for 10-30 min at 300
to allow procathepsin D to autoactivate.
We assay using the RS6 peptide from Ben Dunn's
lab:
Lys-Pro-Ile-Glu-Phe(4nitro)-Phe-Arg-Leu (80 µM)
in 0.1 M Na formate pH 3.5
at 300
measure initial rates as a decrease in absorbance at 300 nm
Isolation of Human Placental Cathepsin D
The human placental cathepsin D is isolated according to the protocol of Huang et al. (1979) with a few modifications.
All steps were carried out at 4o
C.
Approximately 300 grams of tissue frozen at -70 deg is crushed and then homogenized in a Waring blender in 600 ml 10 mM Tris-HCl pH 7.4, 0.5% Brij 35 containing protease inhibitor cocktail 1,10 phenanthroline, PMSF, and leupeptin (not pepstatin!).
Centrifuge at 10000 rpm for 0.5 hour (GSA rotor 8k rpm)
the supernatant was adjusted to pH 3.7 with 5.7 N HCl
Add sodium acetate crystals to 0.1 M and adjust to pH 5.2 with HCL.
Centrifuge at 10000 rpm for 0.5 hour
Apply the supernatant to a pepstatinyl-agarose column (7-10 ml bed volume) pre-equilibrated with 0.1 M sodium acetate pH 3.5, 0.1% Brij 35, 1 M NaCl.
Wash the column with the equilibration buffer until A280 reaches baseline and elute with 50 mM Tris-HCl, pH 8.1 , 0.1% Brij 35, 1 M NaCl. Active enzyme elutes with the pH change. Slow elution is essential! Fast elution will dilute the enzyme. For the Gilson Rabbit pump use blue/yellow tubing at a setting of 25 (about 0.2 ml /min). Collect 2.5 ml fractions. Pool active fractions.
Dialyze against 0.05 M NaPO4 pH7.0, 0.1 M NaCl, 0.1% Brij35
Apply to DEAE Sephacel (3 ml bed volume) pre-equilibrated in 0.05 M NaPO4 pH7.0, 0.1 M NaCl, 0.1% Brij35.
Collect flow through -
Read A280, assay, run on gel
PREPARATION OF PEPSTATIN AGAROSE
(from Huang et al J. Biol. Chem. 254, 11405-11417 (1979))
Materials:
Aminohexyl agarose (Sigma # A6017) =
AH-agarose
Pepstatin A (Sigma# P4265)
N,N dimethyl formamide (Aldrich Gold, stored
over molecular sieves for drying) = DMF
N,N dicyclohexyl carbodiimide (Aldrich ) = DCC
N hydroxysuccinimide (Aldrich) = NHS
Dioxane (Aldrich Gold, stored over molecular
sieves for drying)
Pepstatin approx 25 mg/mL in 1mL of DMF 36 µMoles
add the following at 2moles/mole of pepstatin
DCC in DMF 72 µMoles 15.1 mg
NHS in DMF 72 µMoles 8.3 mg
DMF to final volume of about 12 ml
mix over night at 4o - We use a Pierce reacti-vial or something like that, remember everything is water free as much as possible! DMF stored over molecular sieve.
AH agarose (5-10 uMoles/ ml) gently suction filter (scintered glass frit M or C) do not dry it out.
weigh out about 20-30gms (wet weight) i.e. about 4 fold molar excess of ligand on the bead
Wash the beads with dioxane to remove water
Resuspend beads in 10 mL of dioxane , then add to activated pepstatin, mix overnight at room temp.
Wash beads in scintered glass funnel with:
1L of 1:1 dioxane:DMF
3L of dd H2O
2L of 50mM Tris pH 8.5, !M NaCL
1L dd H2O
1L of 50mM Na Acetate pH3.5, 1M NaCl
beads are now ready to use
for storage at 4o wash with 2L of 5mM Na Phospate pH 7.5
resuspend in equal volume of 5 mM Na Phosphate and add NaN3 to 0.02%
Note it is difficult to determine the
amount of pepstatin bound per mL of gel. Accurate determination
requires measurement of depletion of pepstatin from the reaction
mixture. Best to test the beads' ability to bind and elute enzyme
activity.