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Removal of N-Terminal His-Tags 239
DAPase test of pGAPase performance
1 2 3 4 5 6 7 8 9 10 11 12
TNFα
ΔTNFα
DAPase
2h DAPase treatment 37°C,
DAPase stop
V R S S S R T P S D
TNFα
ΔTNFα
Fig. 4. DAPase test for pyroglutamyl removal by pGAPase (see Subheading 3.5.).
DAPase treatment of tumor necrosis factor (TNF) results in cleavage only if
pyroglutamyl has been removed by pGAPase. Thus, addition of DAPase results in the
cleavage of the first six amino acids resulting in truncated TNF ( TNF). DAPase
cleavage stalls at TP (3,4).
2. Collect the flow-through (measured by absorbance at 280 nm) and pool fractions
containing pyroglutamyl-TNF protein.
3. Prepare a pGAPase mixture: Mix 75 units pGAPase (3 ml) and 300 μl 20 mM
cysteamine and incubate 5–10 min at room temperature. See Note 9 for ratios
between pGAPase and target proteins.
4. Add pGAPase to the pooled sample.
5. Incubate at 37°C for 1 h (no mixing required).
a. After the pGAPase reaction, the mixture is passed through a 5-ml HisTrap
column as above to remove pGAPase.