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Removal of N-Terminal His-Tags 235
be chosen for the Gln residue, and the first amino acid of the mature target
protein must immediately follow. Alternatively, TAGZyme pQE-1 is the vector
of choice whenever the sequence of the protein allows cloning into the bluntended
PvuII restriction site, which links the first amino acid of the target protein
to the Gln stop point (see Fig. 2).
2.2. The Model Protein: Human TNF
Tumor necrosis factor (TNF) is a multifunctional pro-inflammatory
cytokine with effects on lipid metabolism, coagulation, insulin resistance and
endothelial function (8). We have used TNF as a model to illustrate the
properties of TAGZyme. Similar approaches can be adapted for other proteins.
The N-terminal sequence of mature TNF is VRSSSRTPSD. The sequence of
the His-tag of the recombinant TNF used here is shown in Fig. 1A. Basically,
the sequence includes the UZ-HT15 His-tag (4) with the additional Gln residue
adjacent to the first residue (V) of TNF.
2.3. Initial IMAC Purification of His-Tag protein from E. coli
An E. coli strain containing a plasmid that carries the sequence of human
TNF as a fusion with the UZ-HT15 His-tag sequence (see Fig. 1) was used.
This strain was cultured in shake flasks (600 mL) essentially as described
in ref. 4. Briefly, the strain was grown to an OD 600 nm between 0.4 and 0.6.
Gene expression was induced by addition of 0.5 mM Isopropyl 1-thio--Dgalactopyranoside
(IPTG). Cells were harvested after 4–5 h of induction.
2.3.1. Buffers
1. Lysis buffer: 25 mM Tris–HCl, 300 mM NaCl, pH 8.
2. Buffer A: 20 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM imidazole, pH 7.5.
3. Buffer B: 20 mM NaH 2 PO 4 , 300 mM NaCl, 1 M imidazole, pH 7.5 (see Note 3).
4. Buffer C: 20 mM sodium phosphate, 150 mM NaCl, pH 7.0.
5. Buffer D: 20 mM sodium phosphate, 150 mM NaCl, 2 mM cysteamine, pH 7.0.
2.3.2. Step A
IMAC Stationary Phase: Ni-Chelating Sepharose 6 FF column (2 cm 2 ×
6 cm).
1. Preparation of Ni-chelating Sepharose 6 FF is performed according to the method
described by the manufacturer.
2. Lysis treatment: Lysozyme (30 mg/ml; Sigma) and Benzonase (250 units/μL;
Merck) in lysis buffer.
3. Buffer A for wash and buffer B for elution.