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Removal of N-Terminal His-Tags 237
pGAPase removal of the N-terminal pyroglutamyl residue has been effectively
performed. The truncated TNF displays a different migration that
is detectable by sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS–PAGE). If pyroglutamyl has not been removed from the N terminus
during DAPase/Qcyclase treatment, then DAPase will not cleave and no size
alteration will be observed on this test. Thus, DAPase treatment can be used
as a diagnostic method to test the efficiency of pyroglutamyl removal by
pGAPase.
3. Methods
3.1. Initial IMAC Purification of His-Tag Protein from E. coli
1. Harvest cells from 2 × 600 ml culture by centrifugation (15 min, 4°C, 5000 × g)
and resuspend in 80 ml pre-cooled lysis buffer.
2. Freeze/thaw the cell pellets to aid cell lysis and add 60 mg lysozyme (2 ml; 30
mg/ml) and 1250 units benzonase (5 μl; 250 units/μl). Incubate for 1hat4°C
(no mixing required) and centrifuge for 30–45 min (4°C, 13,000 × g).
3. Apply the sample (∼80 ml) at a flow rate of 2 ml/min onto a Ni-chelating
Sepharose 6 FF column (2 cm 2 × 6 cm) pre-equilibrated with lysis buffer at 4°C.
4. Wash the column with 20 ml lysis buffer at a flow rate of 2 ml/min.
5. Wash the column with 50 ml buffer A at a flow rate of 2 ml/min.
6. Elute the His-tag protein using a linear gradient (80 ml) from buffer A to buffer
B at a flow rate of 1 ml/min, collecting 2 ml fractions (see Note 3).
7. Run diagnostic SDS–PAGE/activity assay with the obtained fractions to identify
fractions containing the His-tag protein (see Fig. 3A).
8. Pool fractions containing the purified His-tag protein.
9. Apply the pooled fractions of the purified His-tag protein (typically 30–40 ml)
at a flow rate of 4–5 ml/min onto a Sephadex G25 F column (5.3 cm 2 ×30cm)
equilibrated with buffer C.
10. Wash the column with buffer B at a flow rate of 4–5 ml/min and collect the
desalted His-tag protein (measured by absorbance at 280 nm) in one pool.
11. Measure the protein concentration and proceed to removal of the tag.
3.2. Removal of the Tag, Step 1: DAPase and Qcyclase Treatment (for
∼80 mg protein)
1. Prepare a DAPase/Qcyclase mixture:
a. Mix 2 units DAPase (200 μl) and 10 μl 20 mM cysteamine and incubate
5–10 min at room temperature (see Notes 5 and 6).
b. Add 240 units Qcyclase (4.8 ml). For information on the required buffer pH,
see Note 7. For information on reducing enzyme needs see Note 8.