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Preparation, Analysis and Use of an Affinity Adsorbent 129
mix for 15 min. Pass the solution through Whatman no. 1 filter paper (approximate
pore size of 11 μm) to remove undissolved dithiodipyridine. Perform a mass
balance to determine the molar concentration of the reagent (see Note 9).
2. Add 1 ml of reagent to 1 ml of a 50% slurry of adsorbent in DI water (see Note 10).
3. Incubate the solution on a rotary stirrer for 1hatroom temperature.
4. Measure the optical density of the supernatant at 343 nm (OD 343 nm ) and compare
the results against a calibration curve created using a serial dilution of pure reduced
glutathione in DI water (0.1 g/ml to 5×10 −6 g/ml is a good starting point).
3.4. Preparation of Clarified Lysate Containing GST–ZnF Via
Freeze/Thaw Lysis
1. BL21 Escherichia coli cells containing the expressed fusion protein are harvested
from fermentation medium by centrifugation at 5000 × g (5300 rpm in a Beckman
JA-10 centrifuge) for 10 min in a room temperature rotor (see Note 11).
2. Resuspend the cell pellet in PBS buffer and lyse the cells by six cycles of
freeze/thaw lysis (place sample in liquid nitrogen until completely frozen, then
place sample in 37°C water bath until completely thawed).
3. Clarify the lysis solution by centrifuging at 15,000 × g (9200 rpm in a Beckman
JA-10 centrifuge) for 15 min, then syringe the supernatant through a 0.22-μm
filter. The expressed GST–ZnF remains soluble in the liquid fraction, so no further
processing or refolding is required. Prepare clarified lysate on the day it is to be
used.
3.5. Preparation of Unclarified Lysate Containing GST–ZnF Via
Homogenization
1. Load room temperature cell culture into the homogenizer holding unit.
2. Pump the culture through the ceramic homogenizing valve at an operating pressure
of 1000 bar (see Note 12).
3. Immediately hold the homogenized product on ice until the temperature returns to
room temperature. Repeat the procedure a further two times. Prepare homogenized
cell lysate on the day it is to be used (see Note 13).
3.6. Packed Bed Chromatographic Protein Purification
1. Pack a chromatography column via gravity settling with the adsorbent prepared
as given in Subheading 3.1.
2. Equilibrate the column with 10 column volumes of PBS buffer.
3. Load GST–ZnF-containing lysate onto the column at an approximate rate of
60 cm/h (see Note 14). The binding capacity of GST–ZnF for the affinity adsorbent
was approximately 6 mg/ml. Hence, a volume of lysate containing approximately
6 mg of GST–ZnF was loaded per ml of adsorbent.