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Removal of N-Terminal His-Tags 241
Table 1
Sequence-Dependent Cleavage Efficiency of DAPase
Rapid Medium Slow No cleavage
Xaa-Arg Asp-Asp ab Gly-Ser Lys-Xaa
His-Gln Glu-Glu ab Ser-Met Arg-Xaa
His–Gly Glu-His b Gly-Met Xaa-Xbb-Pro
Xaa-Lys Gly-Phe c Xaa-Phe c Xaa-Pro
Gly-His Ser-Tyr Gln-Xaa (in the presence
His–Ala Ala-Ala
of Qcyclase)
His–His Phe-Xaa c
His–Met Xaa-Asp b
Ala-His Xaa-Glu b
Met-His
a Medium-to-slow cleavage rate.
b Positively or negatively charged side chains inhibit DAPase cleavage.
c With a few exceptions, slow cleavage rate apply to all dipeptides containing Phe, Ile, Leu,
Tyr and Trp in either of the two positions.
TNFα cleavage at 4°C
M 1 2 3 4 5 6 7 8 9 10 11 12 M
66.3
55.4
36.5
31.0
21.5
14.4
6.0
mU/mg 15 10 5 2.5 1
Fig. 5. DAPase/Qcyclase treatment of histidine tag (his-tag) tumor necrosis factor
(TNF) using lower enzyme amounts and incubation at 4°C. Lane M: molecular
weight marker (in kDa); lanes 1 and 12: untreated His-tag TNF; lanes 2, 4, 6, 8 and
10: 1-h treatment; lanes 3, 5, 7, 9 and 11: overnight treatment. The amount of DAPase
per mg protein is shown.