Molecular Techniques and Methods

Purification of Recombinant Proteins
Tagged with Affinity Handles

Copy Right © 2001/ Institute of Molecular Development LLC


The affinity purification of soluble fusion proteins with affinity tags became relatively simple and predictable. The basic concept is based on the specific interaction of an additional polypeptide tag fused to the target protein with an immobilized ligand. A cell lysate including the fusion protein with the affinity tag is passed through an affinity column containing a ligand that specifically interacts with the affinity handle. The fusion protein is retained by the ligand while the other contaminating proteins can be washed through the column. After elution of the fusion protein, a chemical or enzymatic method is used to cleave the fusion protein at the junction between the two protein moieties. The cleavage mixture is again passed through the column to allow the affinity handle to bind and the target protein is collected in the flow-through fraction.

Metal Chelate Affinity Chromatography (MCAC)
Metal chelate affinity chromatography (MCAC), also known as immobilized metal affinity chromatography (IMAC), is based on the co-ordination of chelated metal ions by accessible amino acid residues such as histidines in the target protein.

Metal affinity separations exploit the affinities for metal ions that are exhibited by functional groups on the surface of proteins. Although a number of functional groups participate in metal binding in metalloproteins, the actual situation for MCAC is less complex. Proteins are retained on metal affinity columns according to the number of accessible surface histidines. However, individual histidyl residues vary in their affinities for immobilized metal ions, depending on the influence of neighbouring residues. Histidine is a relatively rare amino acid, accounting for about 2% of the amino acids in globular proteins. Only about half of the histidine residues are exposed on the protein surface. Proteins that contain neighbouring histidines are not common in bacteria. Therefore, target proteins with genetically introduced poly-histidine tails display a high affinity interaction with metal ions and permit efficient purification. The strength of interaction between a histidine-tailed protein and the affinity matrix is dependent on many factors such as length of the histidine tail, the choice of the metal ion, the pH, and the properties of the target protein itself. In most cases, a poly-histidine tail (located either at the N- or C-terminus) does not interfere with the biological activity of the target protein. Therefore, there is often no need to remove the histidine tail after purification of the target protein.

In order to utilize the protein-metal ion interaction for chromatographic purposes, the metal ion must be immobilized on to an insoluble support. This is achieved by attaching a chelating group to the chromatographic matrix. One chelating group used in this technique is iminodiacetic acid (IDA), coupled to a matrix via a long hydrophilic spacer arm. The spacer arm ensures that the chelating metal (for example, Cu2+ or Zn2+) is fully accessible to all available binding sites on a protein. IDA is a tridentate chelator. As the metal ions will co-ordinate four to six ligands, the remaining co-ordination sites are occupied by water molecules or buffer components, which can be displaced by appropriate protein functional groups. Nitrilotriacetic acid (NTA) with four chelating sites has been used in combination with Ni2+.

The most commonly used metals for MCAC are Cu2+, Ni2+, or Zn2+. Protein retention on different metals reflects the affinity of the metal ion for imidazole: both protein retention and stability constants for complexation with imidazole follow the order Cu2+ > Ni2+ > Zn2+. The choice of the best metal is not always predictable. Copper often affords much tighter binding to proteins than does zinc. However, the weaker binding achieved using zinc may be exploited for selective elution of a protein mixture in some cases. Therefore, the appropriate choice of the metal ion may have to be found in a trial and error process.

Binding of the target protein usually occurs in the pH range 7 to 8. As the pKas of surface histidyl residues are generally between 6 and 7, the imidazole nitrogen will be in the unprotonated state coordinating the metal ion. The choice of starting buffer depends on the chelated metal and on the binding properties of the sample molecules. Sodium acetate, sodium phosphate, and tris-acetate are suitable buffers. Tris-HCl tends to reduce binding and should be used when the metal protein affinity is fairly high. Chelating agents such as EDTA or citrate should not be included. 2-Mercaptoethanol (up to 10 mM) does not interfere with the purification procedure. However, stronger reducing agents such as dithiothreitol should be avoided because the metal ions will be reduced.

The target protein can be eluted from the affinity matrix by three main procedures:
A pH gradient.
Since the histidine imidazole nitrogen co-ordinates metal ions in the unprotonated state, a decrease in pH is sufficient to elute proteins. As several proteins may bind to the gel, optimal purification is achieved with a decreasing pH gradient, normally in the range between pH 7 to pH 4.

A competitive ligand.
Imidazole competes with the protein ligands for the metal ions. Good separations are obtained by eluting with an increasing concentration gradient of imidazole (0-500 mM).

A chelating agent.
Chelating agents such as EDTA or EGTA will strip the metal ions from the gel and cause the elution of all adsorbed proteins. However, this method does not resolve different proteins.

The individual binding properties of histidine-tailed target proteins allow the application of a wide variety of elution conditions. Lowering the pH to 6.0 in a wash step before elution may eliminate contamination, but leave the target protein bound. If it is desirable to keep the pH above 7.0 at all times, the column can be washed with buffer containing up to 40 mM imidazole before application of the competitive ligand gradient. For optimizing elution conditions, the imidazole and pH gradient may be combined.

The binding of histidine-tagged proteins to the resin does not require any functional protein structure and is thus unaffected by strong denaturants such as guanidine hydrochloride or urea. This allows protein also to be efficiently purified from solubilized inclusion bodies.

Examples of Fusion Systems used to Facilitate Purification of Soluble Proteins.
Some systems such as the glutathione S-transferase and maltose binding protein fusions require a correctly folded affinity tag, excluding purification under denaturing conditions. In contrast, poly-histidine tagged fusion proteins can be recovered under native and denaturing conditions.

Purification Tag
Eluted by
Poly-Arginine5 5 aa Ion-exchange chromatography Salt gradient
Poly-Aspartic acid5-16 5-16 aa Ion-exchange chromatography Salt gradient
Glutamic acid 1 aa Ion-exchange chromatography
Poly-Histidine2-6 2-6 aa Metal chelate affinity chromatography Low pH/ Imidazole/ EDTA
Poly-Cysteine4 4 aa Thiopropyl Cysteine and DTT
Poly-Phenylalanine11 11 aa Phenyl Ethylene glycol
RecA 144 aa Anti-RecA antibody pH 2.5
c-myc 11 aa 9E10 pH 3.0
FLAG 8 aa Anti-FLAG M1/ Anti-FLAG M2 pH 3.5
beta-Galactosidase 116 kDa p-aminophenyl-beta-D-thiogalactopyranoside Borate buffer (pH 7.0)
Glutathione s-transferase 26 kDa Glutathione Glutathione
Chloramphenicol acetyltransferase 24 kDa Chloramphenicol Chloramphenicol
Staphylococcal Protein A 31 kDa Immonuglobulin G (IgG) pH 3.5
ZZ (Synthetic protein A analogue) 14 kDa Immunoglubulin G (IgG) pH 3.4
Streptococcal Protein G 30 kDa Albumin pH 2.8
Maltose Binding Protein 40 kDa Amylose Maltose




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