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.
* Estimate for a (His)6 fusion protein of Mr 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.
Figure 49. Puriﬁcation of egg white proteins on HiTrap® Chelating HP 1 mL, using the metal ion Cu2+.
|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|
|Elution:||8 mL linear gradient 0-100% elution buffer|
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.
The following guidelines may be used for preliminary experiments to select the metal ion that is most useful for a given separation:
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.
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.
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. 5010 repetitive puriﬁcations of GST-(His)6 without reloading the column with Ni2+ between the runs.
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.
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).
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.
Stable in all commonly used aqueous buffers and denaturants such as 6 M guanidine hydrochloride, 8 M urea and other chaotropic agents.
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.
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