10
diameter; e.g., Bio-Rad Econo-Pac 0.7 × 5.0- cm), and let the
liquid drain. Sequentially rinse the resin with 5 bed volumes of
Milli-Q water, 2 bed volumes of NiSO 4 solution, 5 bed volumes
of Milli-Q water, and 5 bed volumes of bacterial lysis buffer.
Let the column drain by gravity flow between each rinse. Do
not let the resin dry. Keep the column at 4 °C. Following these
steps, the column is ready to use (see Note 10).- Filter the lysate with a 0.45 μm Durapore Stericup HV filter
unit. Rinse the filter with 5 mL of bacterial lysis buffer and
pool with lysate (see Note 11). - Apply the lysate to the column and let drain by gravity flow.
- Wash the resin five times with 10 mL of washing buffer. Let
the liquid drain between each wash. - Re-equilibrate the column with 5 bed volumes of bacterial lysis
buffer, and leave ~1 mL of buffer on top of the resin to prevent
air bubbles from entering the resin. - Attach the gradient maker (valve closed) to the pump, the
pump to the column flow adaptor, the adaptor to the column,
and the column outlet to the fraction collector. Ensure that all
tubing is full of bacterial lysis buffer. - Add 12.5 mL of bacterial lysis buffer in compartment 1 and
12.5 mL of elution buffer in compartment 2 of the gradient
maker. Set the pump flow rate to 1 mL/min, open the gradi-
ent maker valve, and collect 1 mL fractions. Continuously stir
compartment 1. Keep all fractions on ice (see Note 12). - Just before the end of the gradient, add 5 mL of elution buffer
into compartment 2 of the gradient maker. This step will elute
any remaining His-tagged protein. - Measure the absorbance of each fraction at 280 nm. Analyze
10 μL of every other fraction by SDS-PAGE (see Note 13). - Pool the purest and most concentrated fractions. Measure the
absorbance of the pooled fractions at 280 nm, and estimate
the initial caspase concentration using the Edelhoch relation
(see Note 14). - Prepare 50–100 μL aliquots and freeze at −80 °C (see Note 15).
- Perform an active site titration of the caspase preparation
according to Subheading 3.2.2.
Full-length caspase-8 does not express as a soluble protein in E. coli,
it is exclusively found in inclusion bodies. Therefore, a purification
strategy that employs denaturants to solubilize the caspase is
required. The protein must then be refolded to recover the enzy-
matic activity. The following procedure allows for the production of
small but enzymatically pure and active preparations of full- length
caspase-8 (adapted from a protocol from Christina Pop, personal
communication). The first purification step involves the preparation3.1.2 Protocol for
Full-Length Caspase-8
Production in E. coli
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