Immobilized metal ion affinity chromatography (IMAC) is based on the specific, reversible coordinate interactions between amino acids, particularly histidine, and metals in aqueous solutions. This technique has been widely used to purify polyhistidine-containing proteins or peptides by passing the His-tagged protein or peptide through in a column containing immobilized metal ions so that the protein or peptide is retained because of its affinity to the metal ion. The target protein or peptide can then be eluted from the column by lowering the pH or by adding a competitive molecule, such as imidazole in most common practice.
The property and performance of each IMAC resin format is determined by the combination of three components of the resin: the matrix support, the chelate ligand that is used to functionalize the matrix surface, and the immobilized metal ion that is in complex with the ligand.
The matrix supports being widely used in research and scale up processing applications include 4% agarose or highly crosslinked 6% agarose (6 fast flow). Various polymer based matrices and magnetized matrices have been also developed mainly for applications where only small amounts of proteins are needed.
Current commercially available chelate ligands include nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), as well as pentadentate ligands, which vary in available valencies for interaction with the metal ion and therefore result in various chelate-ion complexes with different degrees of stability. Chelate ligand NTA is well suited for the purification applications, and thus are the most commonly used. Pentadentate ligands, such us our proprietary ligand on Ni-Penta™ resins, offering by far the most stable, chemical-tolerant immobilization of metal ions.
The metal ions that most often used for purification of His-tagged proteins are divalent cations, including copper (Cu2+), nickel (Ni2+), zinc (Zn2+), and cobalt (Co2+).
Combinations of these three variables (matrix, ligand, and ion) provide a collection of IMAC resins that we can choose from for our specific purification needs. Choosing the appropriate IMAC resin format contributes to the success of obtaining desired amount of pure target His-tagged protein for downstream applications.
|Resin Matrix Supports||Chelate Ligands||Immobilized Metal Ions|
6% highly cross-linked agarose
PENTA (our proprietary pentadentate ligand)
|Cu2+, Ni2+, Zn2+, and Co2+|
When selecting the IMAC resin, we first need to consider the following properties of the resin:
- Binding capacity of the resin, which impacts the yield of target proteins
- Bind specificity of the resin, which influences the purity of target proteins
- Tendency of metal leaching from the matrix
- Chemical stability of the resin, which impacts the feasibility of resin being adapted in the optimal buffer conditions for the protein
- Methodology of separation in the application : manual (by batch/gravity flow) or automation (by pressurized flow)
Let’s look into each of these characteristics.
Binding capacity and inversely associated specificity
The binding capacity and the specificity of an IMAC medium are always the two sides of the coin, as they respectively determine the yield and purity of the final purified protein. These two characteristics of a certain type of resin are impacted by the immobilized ion as well as the functional ligand, which the ion is in complex with.
The divalent cations, Cu2+, Ni2+, Zn2+ and Co2+, show differential binding affinity in inverse association with the specificity for the His-tag as illustrated in Figure 1. In practice, the choice of metal ions is often highly dependent on the purification application. The ions most widely used for purifying his-tagged proteins are nickel ion (Ni2+) for high yield purification applications and cobalt ion (Co2+) for applications that require higher purity, respectively. In some special applications, ions copper (Cu2+) or zinc (Zn2+) may be more suitable. For example, ion Cu2+ has greater affinity and the copper-immobolized resins have been used to enrich low abundant proteins from crude lysate, prior to selective purification with Cobalt IMAC for superior purity.
Figure 1. Inverse association of affinity/capacity and specificity of metal ions for His-tag.
The chelate ligand, which is utilized to functionalize the matrix support for immobilization of the metal ion, also has a significant contribution to the affinity and specificity properties of the IMAC medium. Current mostly used chelate ligands to immobilize ions on the matrix support is NTA. The NTA ligand coordinates the Ni2+ with four valencies (tetradentate, coordination number 4), and two valencies are available for interaction with imidazole rings of histidine residues. This ratio seems to be most effective for purification of His-tagged proteins. IDA coordinates a divalent ion with three valencies (tridentate, coordination number 3), leaving three valencies ready for imidazole ring interaction. The use of IDA ligand allows greater His-tag binding capacity, in comparison with NTA. At Marvelgent, besides NTA and IDA, we also offer Ni-Penta™ resins that utilize a proprietary pentadentate ligand. The pentadentate ligand stably immobilizes the nickel ion on the matrix support with five coordination sites. Therefore, the binding between the nickel ion and the agarose support is so strong that it will not be disrupted by high concentration of many commonly used chemicals, including EDTA, DTT and β-ME at higher concentrations. On the other hand, however, because only one valency is available for interaction with histidine residue, Ni-Penta resins have higher selectivity but lower capacity for His-tagged proteins (Read more on Ni-Penta properties contributing to high selective purification of His-tagged proteins ...).
Illustrated in Figure 2 are the structures of these functional ligands in complexes with the nickel ion.
Figure 2. Structural illustration of three types functionalized agarose matrices pre-charged with nickel ions. The polydentate ligands, IDA, NTA, and our proprietary pentadentate chelating ligand, respectively provide 3 time, 4 times, and 5 times bonding sites for the nickel ion. The stability of Ni-bonds determines their binding capacities as well as the levels of their retainable affinity for polyhistidine in various solution conditions.
Chemical stability of the IMAC medium to be used is often the critical factor that limits the conditions from which the target protein can be purified. The following table lists several protein properties and conditions that often lead to challenges of obtaining the pure, intact, functional protein of interest.
|Conditions raised from the nature of the target protein|
|Large molecular size||The bulky size of the protein constrains binding affinity to the metal ion.||Purify with higher binding capacity IMAC resins, eg. Ni-IDA. A following chromatography with gel filtration to remove lower molecular weight contaminants may be necessary.|
|Containing post-translational modifications||Modifications may lead to weaker binding affinity to the metal ion as the His-tag might not be well-exposed.||Higher binding capacity IMAC resins, eg. Ni-IDA or Ni-NTA resins.|
|Presence of Cysteine (SH- bond)||The presence of DTT or β-ME is required.||Ni-Penta™ resin is recommended because of its tolerance to DTT and β-ME.|
|Cysteine-rich and metal ion-reactive||The presence of DTT and EDTA is required.||Ni-Penta™ resin is recommended because of its tolerance to DTT and β-ME.|
|Oxygen-sensitive||The presence of DTT or β-ME is required.||Ni-Penta™ resin is recommended because of its tolerance to DTT and β-ME.|
|Susceptible to proteolysis by Metalloproteases||The presence of EDTA is required throughout purification.||Ni-Penta™ resin is recommended because of its superior tolerance to EDTA.|
|Imidazole-sensitive||Excessive amount of Imidazole cannot be used to compete with Histidine-ion interaction. Minimal usage of imidazole is required in binding, washing, and elution.||Ni-Penta™ and Co-NTA resins are recommended. In the purification protocol using either type of resin, no imidazole is required in the binding buffer, 0~5 mM imidazole in the wash buffer. And a much lower concentration of imidazole is often sufficient to elute the target protein.|
|Conditions resulted from the expression system|
|Membrane-integral protein||The protein can be engaged with nonspecific interactions. Ionic and nonionic detergents may be required.||NTA-functionalized resins generally perform well in various ionic and nonionic detergents. However, if higher strength of detergents is necessary, Ni-Penta™ resins are recommended as they outperform NTA resins in many chemicals for longer period of time.|
|Being secreted from eukaryotic host into culture media||The protocol starts with Large volume of raw material containing EDTA, β-ME, which are incompatible with NTA and IDA. Removing these agents by dialysis may involve a lot of tedious sample handling.||Ni-Penta™ resins are recommended as they retain high selectivity in presence of EDTA, DTT, and β-ME.|
|Low-abundance expression||Lower concentration of the target protein in solutions results in weaker affinity and slower binding kinetics.||Higher binding capacity IMAC resins (eg. Ni-IDA) can help enrich the protein. If high purity of the final product is required, it can be followed by high selective capture with Co-NTA or Ni-Penta resins.|
Potential leaching of the metal ion from the matrix depends on the stability of the chelate-ion complex. Stronger bonds between the chelating ligand and the ion, the lower risk of metal leakage (ie. the Ni-Penta complex). Whereas, weaker bond (as in the Ni-IDA, Ni-NTA, and Co-NTA complexes) inevitably leads to higher leaching tendency. Among IMAC resins, the lowest metal leaching can be obtained if a pentadentate ligand is used, which coordinates the ions tightly, and the higher ion leakage is observed with IDA ligand.
Another consideration is whether there is any toxic effect caused by the ion leaching into the eluate and should be avoided in the downstream applications. For example, as a heavy metal, nickel ion is a carcinogen, and may not be the metal of choice in some purification applications. In such cases, Co-NTA resins or anti-strip, chemical-stable Ni-Penta resins may be more desirable.
Rigidity and mechanical stability of the matrix support
The next question to ask is which purification method will be used, a manual purification or an automated purification. Non-crosslinked agarose is a flexible, versatile matrix support for IMAC, providing a better binding capacity in general. Whereas highly-crosslinked agarose offers the rigidity and mechanical stability that is prerequisite for purification on an automatic system, such as AKTA system.
In conclusion, metal ions and the chelate ligands exhibit various degrees of affinity/capacity and specificity/selectivity for His-tag. Therefore, by combining different ions and ligands on the matrix support, an array of IMAC resins with differential properties and performance in the purification of His-tagged proteins provide a selection spectrum for various specific applications. Proper use of the matching IMAC resin warrants the success of obtaining sufficient amount of target His-tagged protein with desired purity for downstream applications.
At Marvelgent, we offer a wide range of IMAC resins for various purification needs, from fast research laboratory separation to preparative and process scale applications. Contact our scientists for suggestions and technical supports.