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238 Arnau et al.
Fig. 3. Immobilized Metal Affinity Chromatography (IMAC) purification of
histidine tag (His-tag) tumor necrosis factor (TNF) in Escherichia coli
(A, see Subheading 3.1.) and subsequent tag removal using TAGZyme
(B, see Subheadings 3.2. and 3.3.). (A) Lane M: molecular weight markers (sizes
in kDa); lane 1: crude extract; lane 2: crude extract after centrifugation; lane 3: flow
through from the IMAC column (see Subheading 3.1.); lanes 4–11: eluted fractions
24, 26, 28, 30, 32, 34, 36 and 38, respectively. (B) Cleavage of His-tag TNF obtained
from the initial IMAC. Lane 1: His-tag TNF; lane 2: DAPase/Qcyclase 10-min
treatment; lane 3: DAPase/Qcyclase 20-min treatment; lane 4: DAPase/Qcyclase 30-
min treatment; lane 5: eluted tag-free TNF after pGAPase treatment and subsequent
elution from IMAC.
2. Add the 5 ml DAPase/Qcyclase mixture to the desalted sample of His-tag protein
(typically 35–50 ml, in the example shown containing ∼80 mg for TNF).
3. Incubate (no mixing required) at 37°C for 30 min. At time 10, 20 and 30 min, take
25 μl aliquots to follow the cleavage of the His-tag and mix with 2× SDS–PAGE
sample buffer containing Dithiothreitol (DTT) (see Fig. 3B). See Note 8 for the
use of lower DAPase amounts.
3.3. Removal of the Tag, Step 2: Removal of DAPase and Qcyclase
Using Subtractive IMAC Followed by Removal of Pyroglutamyl Using
pGAPase (∼80 mg protein)
1. After the DAPase/Qcyclase reaction, the enzyme reaction mixture is passed
through a 5-ml HisTrap column to remove DAPase, Qcyclase, unprocessed Histagged
TNF and other unspecific IMAC binders using a flow rate of 2 ml/min.
This step is called subtractive IMAC because the primary role is the removal
(“subtraction”) of His-tag proteins (DAPase and Qcyclase together with poorly
processed protein molecules resulting from, e.g., removal of initial Met during
expression) and to elute the tag-free protein.