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236 Arnau et al.
2.3.3. Step B
Buffer exchange on Sephadex G25 F (see Note 4) stationary phase: Sephadex
G25 column (5.3 cm 2 × 30 cm) equilibrated with buffer C.
Cysteamine–HCl and Imidazole were obtained from Sigma. Sephadex G-25
F and Ni-chelating Sepharose 6 FF.
2.4. DAPase and Qcyclase Treatment
DAPase (10 units/ml) and Qcyclase (50 units/ml).
2.5. Removal of DAPase and Qcyclase Followed by Removal
of Pyroglutamyl Using pGAPase and Subtractive IMAC
Stationary support: Freshly prepared HisTrap column (1 ml) and equilibrated
with pGAPase (25 units/ml, Qiagen) prepared in buffer C.
2.6. Subtractive IMAC Using On-Column-Bound pGAPase
Stationary supports:
1. Column 1: 5 ml freshly prepared HisTrap equilibrated with 25 ml buffer C.
2. Column 2: 5 ml HiTrap equilibrated with buffer C.
3. Column 3: 20 ml pGAPase-chelating Sepharose FF (50 units/ml) is prepared by
the following method at room temperature:
a. 20 ml chelating Sepharose FF packed in a2cm 2 × 20-cm column is loaded
with 200 ml 10 mM ZnSO 4 pH 7 at a flow rate of 2 ml/min.
b. Wash the column (2 ml/min) with 40 ml H 2 O.
c. Equilibrate (2 ml/min) with 30 ml buffer C.
d. Load the Zn-chelating Sepharose FF column (2 ml/min) with 1000 units
pGAPase in 200 ml buffer C.
e. Mix the contents in the column to ensure a homogeneous material and pack
the column again.
f. Equilibrate (2 ml/min) with 30 ml buffer D (see Note 5).
g. Equilibrating (2 ml/min) with 60 ml buffer C.
4. Chelating Sepharose, HisTrap and HiTrap were from GE Healthcare.
2.7. DAPase Test for Pyroglutamyl Removal in TNF by pGAPase
After removal of pyroglutamyl by pGAPase and production of a tag-free
protein, the first dipeptides of TNF (ValArg SerSer SerArg) can be further
processed before a stop position is encountered (ThrPro) if DAPase is added
(see Fig. 1A). Thus, treatment of tag-free TNF using DAPase for 2 h (as
described in Subheading 3.5.) would result in a truncated TNF only if