Immobilized Metal Chelate Affinity Chromatography (IMAC)

• Purification Example
• Development of a Separation Protocol
• Selecting the Metal Ion
• Choice of Binding Buffer
• Choice of Elution Buffers
• Scale of Operation
• Cleaning
• Chemical Stability
• Storage
• Related Products

Proteins and peptides that have an affinity for metal ions can be separated using metal chelate affinity chromatography. The metals are immobilized onto a chromatographic medium by chelation. Certain amino acids, e.g. histidine and cysteine, form complexes with the chelated metals around neutral pH (pH 6–8) and it is primarily the histidine-content of a protein which is responsible for its binding to a chelated metal.

Metal chelate affinity chromatography is excellent for purifying recombinant (His)₆ fusion proteins (page 47, Poly (His) fusion proteins) as well as many natural proteins. Chelating Sepharose™, the medium used for metal chelate affinity chromatography, is formed by coupling a metal chelate forming ligand (iminodiacetic acid) to Sepharose™.

Before use the medium is loaded with a solution of divalent metal ions such as Ni²⁺, Zn²⁺, Cu²⁺, Ca²⁺, Co²⁺ or Fe²⁺. The binding reaction with the target protein is pH dependent and bound sample is, most commonly, eluted by reducing the pH and increasing the ionic strength of the buffer or by including EDTA or imidazole in the buffer. The structure of the ligand, iminodiacetic acid, is shown in Figure 48.

Figure 48. Partial structure of Chelating Sepharose™ High Performance and Chelating Sepharose™ Fast Flow.

Metalloproteins are not usually suitable candidates for purification by chelating chromatography since they tend to scavenge the metal ions from the column.

Purification Options

	Binding capacity	Maximum operating flow	Comments
His MicroSpin Purification Module	100 µg/column	Not applicable	Ready to use, prepacked columns, buffers and chemicals for purification of (His)6 fusion proteins.
HiTrap® Chelating HP 1 mL	12 mg/column	4 mL/min	Prepacked column, ready to use.
HiTrap® Chelating HP 5 mL	60 mg/column	20 mL/min	Prepacked column, ready to use.
HisTrap™ Kit	12 mg/column*	4 mL/min	Ready to use, prepacked columns,  buffers and chemicals for purification of (His)6 fusion proteins for up to 12  purifications using a syringe.
Chelating Sepharose™ Fast Flow	12 mg/mL medium	400 cm/h**	Supplied as suspension for packing  columns and scale up.

* Estimate for a (His)₆ fusion protein of M_r 27 600, binding capacity varies according to specific protein.
** See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.

Purification Example

Figure 49. Purification of egg white proteins on HiTrap^® Chelating HP 1 mL, using the metal ion Cu^2+.

Sample:	200 µL egg white (10% in binding buffer filtered through a glass filter)
Column:	HiTrap® Chelating HP, 1 mL, Cu2+-loaded according to the instructions
Binding buffer:	0.02 M sodium phosphate, 1 M NaCl, pH 7.2
Elution buffer:	0.02 M sodium phosphate, 1 M NH4Cl, pH 7.2
Flow:	0.5 mL/min
Elution:	8 mL linear gradient 0-100% elution buffer

Development of a Separation Protocol

Details of a specific purification protocol are given on page 50 (His MicroSpin Purifcation Module, HisTrap™ Kit, HiTrap^® Chelating HP, Chelating Sepharose™ Fast Flow - Performing a separation). This protocol can be used as a base from which to develop purification methods for other proteins and peptides with affinity for metal ions, as shown in Figure 49.

Reuse of purification columns depends on the nature of the sample and should only be considered when processing identical samples to avoid cross contamination.

Selecting the Metal Ion

The following guidelines may be used for preliminary experiments to select the metal ion that is most useful for a given separation:

• Cu²⁺ gives strong binding and some proteins will only bind to Cu²⁺. Load solution equivalent to 60% of the packed column volume to avoid leakage of metal ions during sample application. Alternatively, the medium can be saturated and a short secondary uncharged column of HiTrap^® Chelating HP or packed Chelating Sepharose™ Fast Flow should be connected in series after the main column to collect excess metal ions.
• Zn²⁺ gives a weaker binding and this can, in many cases, be exploited to achieve selective elution of a protein mixture. Load solution equivalent to 85% of the packed column volume to charge the column.
• Ni²⁺ is commonly used for poly (His) fusion proteins. Ni²⁺ solution equivalent to half the column volume is usually sufficient to charge the column.
• Co²⁺ and Ca²⁺ are also alternatives.

Charge the column with metal ions by passing through a solution of the appropriate salt through the column, e.g. 0.1 M ZnCl₂, NiSO₄ or CuSO₄ in distilled water. Chloride salts can be used for other metals.

Several methods can be used to determine when the column is charged. If a solution of metal salt in distilled water is used during charging, the eluate initially has a low pH and returns to neutral pH as the medium becomes saturated with metal ions. The progress of charging with Cu²⁺ is easily followed by eye (the column contents become blue). When charging a column with zinc ions, sodium carbonate can be used to detect the presence of zinc in the eluate. Wash the medium thoroughly with binding buffer after charging the column.

Choice of Binding Buffer

A neutral or slightly alkaline pH will favor binding. Tris-acetate (0.05 M), sodium phosphate (0.02–0.05 M) and Tris-HCl (0.02–0.05 M) are suitable buffers. Tris-HCl tends to reduce binding and should only be used when metal-protein affinity is fairly high.

High concentrations of salt or detergents in the buffer normally have no effect on the adsorption of protein and it is good practice to maintain a high ionic strength (e.g. 0.5–1 M NaCl) to avoid unwanted ion exchange effects.

Chelating agents such as EDTA or citrate should not be included, as they will strip the metal ions from the medium.

Choice of Elution Buffers

Differential elution of bound substances may be obtained using a gradient of an agent that competes for either the ligand or the target molecules. An increased concentration of imidazole (0–0.5 M), ammonium chloride (0–0.15 M), or substances such as histamine or glycine with affinity for the chelated metal can be used. The gradient is best run in the binding buffer at constant pH.

Since pH governs the degree of ionization of charged groups at the binding sites, a gradient or step-wise reduction in pH can be used for non-specific elution of bound material. A range of pH 7.0–4.0 is normal, most proteins eluting between pH 6.0 and 4.2. Deforming eluents such as 8 M urea or 6 M guanidine hydrochloride can be used.

Elution with EDTA (0.05 M) or other strong chelating agents will strip away metal ions and other material bound. This method does not usually resolve different proteins.

If harsh elution conditions are used, it is recommended to transfer eluted fractions immediately to milder conditions (either by collecting them in neutralization buffer or by passing directly onto a desalting column for buffer exchange (page 133, Buffer exchange and desalting).

The loss of metal ions is more pronounced at lower pH. The column does not have to be stripped between consecutive purifications if the same protein is going to be purified, as shown in Figure 50.

Samples:	2.5 mL cell extract containing expressed GST-(His)6
Binding buffer:	20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4
Elution buffer:	20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4
Flow:	2 mL/min, 312 cm/h
Note:	No Ni2+ re-loading of the column between the runs

Figure. 50 10 repetitive purifications of GST-(His)₆ without reloading the column with Ni²⁺ between the runs.

Result: Run No.	Eluted GST-(His)6, total mg
1	2.76
2	2.82
3	2.83
4	2.72
5	2.71
6	2.65
7	2.64
8	2.63
9	2.54
10	2.59

Although metal leakage is very low, the presence of any free metal in the purified product can be avoided by connecting an uncharged HiTrap^® Chelating HP column in series after the first column and before the protein is eluted. This column will bind any metal ions removing them from the protein as it passes through the second column.

Scale of Operation

To increase capacity use several HiTrap^® Chelating HP columns (1 mL or 5 mL) in series (note that back pressure will increase) or, for even larger capacity, pack Chelating Sepharose™ Fast Flow into a suitable column (Appendix 3, Column packing and preparation).

Cleaning

Remove metal ions by washing with 5 column volumes 20 mM sodium phosphate, 0.5 M NaCl, 0.05 M EDTA, pH 7.4.

Remove precipitated proteins by filling the column with 1 M NaOH and incubate for 2 hours. Wash out dissolved proteins with 5 column volumes of water and a buffer at pH 7.0 until the pH of the flow-through reaches pH 7.0.

Alternatively wash with a non-ionic detergent such as 0.1% Triton X-100 at +37 °C for 1 min. Remove lipid and very hydrophobic proteins by washing with 70% ethanol, or with a saw-tooth gradient 0%–30%–0% isopropanol/water.

Media Characteristics

	Composition	Metal ion capacity	pH stability*	Mean particle size
Chelating Sepharose™ High Performance	Iminodiacetic acid coupled to Sepharose™ High Performance via an ether bond.	23 µmoles Cu2+/mL	Short term 2–14 Long term 3–13	34 µM
Chelating Sepharose™ Fast Flow	Iminodiacetic acid coupled Sepharose™ Fast Flow via a spacer arm using epoxy coupling.	22–30 µmoles Zn2+/mL	Short term 2–14 Long term 3–13	90 µM

Chemical Stability

Stable in all commonly used aqueous buffers and denaturants such as 6 M guanidine hydrochloride, 8 M urea and other chaotropic agents.

Storage

Wash media and columns with 20% ethanol at neutral pH (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.

Before long term storage, remove metal ions by washing with five column volumes 20 mM sodium phosphate, 0.5 M NaCl, 0.05 M EDTA, pH 7.4.

The column must be recharged with metal ions after long term storage.