Purification of His-Tagged Proteins

Anne Spriestersbach; Jan Kubicek; Frank Schäfer1; Helena Block; Barbara Maertens    QIAGEN GmbH, Research and Development, Qiagenstrasse 1, 40724 Hilden, Germany
1 Corresponding author: email address: frank.schaefer@qiagen.com

1 Theory

The purification of recombinant proteins is a prerequisite for a lot of downstream applications such as functional and structural studies. Recombinant proteins are commonly expressed with an affinity tag fused to the N- or C-terminus to facilitate purification and detection. One of the most commonly used fusion tags for recombinant protein expression and purification is the His tag, which contains six or more consecutive histidine residues (Waugh, 2005). Due to its small size (0.84 kDa in the case of a hexahistidine tag) and the fact that it is uncharged at physiological pH, the His tag does usually not affect folding and in most cases does not interfere with the structure or function of the fusion protein (Carson et al., 2007). It has a very low immunogenicity and it is compatible with most downstream applications.

Purification of His-tagged proteins by IMAC is based on the affinity of histidine residues for immobilized metal ions (e.g., Ni2+ or Cu2+). The metal ions are immobilized on chromatographic matrices by a chelating ligand, most commonly nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA). While IDA has only three metal-chelating sites, NTA contains four and hence binds the metal ions more stably resulting in reduced ion leaching (Hochuli, 1989; Block et al., 2009). Elution conditions for IMAC of His-tagged proteins are mild and flexible (100–500 mM imidazole, pH 5.9–4.5, or EDTA).

Since the affinity of the His tag toward the Ni-NTA depends only on its primary structure, His-tagged proteins can be purified under native or denaturing conditions (Hochuli et al., 1987). While many recombinant proteins can be produced in a soluble form in E. coli, a lot of other proteins aggregate to insoluble inclusion bodies when expressed at high levels and can be efficiently purified only using denaturing purification conditions. Strong denaturants such as 8 M urea or 6 M guanidinium hydrochloride are usually used to dissolve protein aggregates for IMAC purification. Generally, the binding of His-tagged proteins to the Ni-NTA matrix improves under denaturing conditions since the His tag is fully exposed and the potential of untagged proteins to co-purify is decreased.

Generally, the level of co-purifying contaminants is higher for samples from eukaryotic expression systems, because of the higher abundance of endogenous proteins containing consecutive histidines or metal-binding motifs. In many instances, contaminants that co-purify can be removed using more stringent binding and washing conditions, for example, increasing imidazole concentration, increasing the salt concentration up to 2 M NaCl, or the addition of low concentrations of detergent (for more information on detergents see Explanatory Chapter: Choosing the right detergent). However, a parameter with a very high impact on the purity of the protein preparation is the ratio of Ni-NTA resin to His-tagged protein. To prevent co-purification of untagged proteins with a certain affinity to Ni-NTA, the binding capacity of the resin should be adjusted to the amount of His-tagged protein in the sample (Structural Genomics Consortium, 2008).

Due to the relatively high affinity and specificity of the His-tag, a single IMAC purification step in most cases results in an efficient purification with a reasonably high purity of the target protein preparation, which is sufficient for many applications. However, in some cases optimization of the purification process is required, especially if poorly expressed proteins are to be purified. In this regard, it has been recently reported that purification yields of very low abundance His-tagged proteins from E. coli lysates can be greatly increased by the removal of the periplasmic material prior to cell lysis (Magnusdottir et al., 2009). Alternative approaches to increasing purity include the use of two affinity tags in a double-tag purification procedure (Cass et al., 2005) or the combination of His-tag removal with a reverse IMAC purification step (Block et al., 2008; see Proteolytic affinity tag cleavage).

Although recombinant His-tagged proteins can be expressed and purified from various expression systems, the following protocols focus on the purification of proteins from E. coli as it is the most widely used expression system. The amounts given in the following protocols usually give good results for the purification of His-tagged proteins showing an expression level of 10–50 mg per liter culture volume. However, due to the scalability of the purification procedure, the protocols can be readily scaled up or down depending on the required amount of target protein and the individual expression level of the His-tagged protein. Moreover, the protocols can be easily adapted to other protein expression systems by adjusting the amount of processed culture volume and the cell lysis method (e.g., replacing lysozyme with 1% Igepal CA-630 for insect and mammalian cells. For an example of a lysis protocol, see Lysis of mammalian and Sf9 cells).

Since it is difficult to provide a general protocol for optimal purification for any His-tagged protein, the protocols provided should be used as guidelines and might need to be optimized for a specific target. The buffers given in the protocols can be supplemented to suit the specific requirements of a protein (for an overview see Block et al., 2009), for example, to stabilize a protein by addition of glycerol, to generate reducing conditions or by providing cofactors. However, due to the incompatibility with Ni-NTA resins, strong chelators such as EDTA can be used only in low concentrations and also strong denaturants (e.g., DTT) and ionic detergents (e.g., SDS) are compatible with IMAC only to a certain extent.

2 Equipment

Refrigerated centrifuge

End-over-end shaker

Disposable gravity flow columns

Micropipettors

Pipettes

Pipettor tips

15-ml conical polypropylene centrifuge tubes

1.5-ml microcentrifuge tubes

3 Materials

Sodium phosphate monobasic (NaH2PO4)

Sodium chloride (NaCl)

Imidazole

Sodium hydroxide (NaOH)

Tris base

Urea

Hydrochloric acid (HCl)

Lysozyme (e.g., Roche, Cat. # 10837059001)

Protease inhibitor cocktail without EDTA (e.g., Roche, Complete, EDTA-free, Cat. # 11836170001)

Benzonase (Novagen, Cat. # 70664) (or RNase A and DNase I)

IMAC resin (e.g., Ni-NTA Agarose, QIAGEN, Cat. # 30210, or Ni Sepharose HP, GE Healthcare, Cat. # 17-5318-01)

3.1 Solutions & buffers