A type of Affinity chromatography which is widely used for primary purification of Histidine tagged recombinant proteins

1. Immobilized-Metal
Affinity
Chromatography
Mahendra G. Jadhao
16 Nov.2015

2. Techniques for protein purification according to
specific properties
Protein property Technique
Charge Ion exchange (IEX)
Size Gel filtration (GF)
Hydrophobicity Hydrophobic interaction (HIC), Reversed
phase (RPC)
Biorecognition (ligand specificity) Affinity (AC)
Charge, ligand specificity or hydrophobicity Expanded bed adsorption (EBA) follows the
principles of AC, IEX or HIC

3. Affinity chromatography separates proteins on the
basis of a reversible interaction between a protein (or
group of proteins) and a specific ligand coupled to a
chromatography matrix.

4. Immobilized Metal Affinity Chromatography
• Metal-Chelate Affinity Chromatography (MCAC), also known as
Immobilized Metal Affinity Chromatography (IMAC), was first successfully
demonstrated in 1975 by Porath and collaborators for human serum
proteins.

5. Principle
• Transition metal ions are used, electron pair acceptors.
e.g. Cu2+, Ni2+, Zn2+, Co2+, Fe3+
• Co-ordination between immobilized metal and electron donor
from protein surface.
• Electron donors (N,S,O) present in chelating compound
attached chromatographic support forms metal chelates,
which can be monodentate or multidentate
Denticity : No. of donor groups in a single ligand that bind to
central atom in coordination complex.
• Remaining metals sites are occupied by water molecules and
are exchanged by electron donor from protein.

6. Chelating Compound Coordination Metal ion
Aminohydroxamic acid bidentate Fe(II)
Salicylaldehyde bidentate Cu(II)
Iminodiacetic acid tridentate Cu(II),Zn(II),Ni(II),
Co(II)
Nitrilotriacetic acid tetradentate Ni(II)
Carboxymethylated
aspartic acid (CM-Asp)
tetradentate Ca(II), Cu(II)
Some chelating compounds in use for immobilization in IMAC

7. Figure 27.1. Model of the interaction between residues in the His tag and the metal ion in tri- (IDA), tetra- (NTA), and pentadentate
IMAC ligands (TED).
Helena Block, Barbara Maertens, Anne Spriestersbach, Nicole Brinker, Jan Kubicek, Roland Fabis, Jörg Labahn, Frank Schäfer
Chapter 27 Immobilized-Metal Affinity Chromatography (IMAC): A Review
Methods in Enzymology, Volume 463, 2009, 439–473
http://dx.doi.org/10.1016/S0076-6879(09)63027-5
Imidazole rings of
Histidine

8. Fig. Schematic illustration of the protein binding to a metal-chelated affinity support.
Strong binding of a protein onto the IMAC matrix is achieved predominately by multi-
point attachment of native or engineered surface histidines Ža., or by histidine tag Žb.
added to the N- or C-terminus of the protein. There are many possibilities for the
construction of efficient His tags considering the number of histidines, their location
and microenvironment.

9. His-Tag for Purification of
Recombinant Proteins
• It has been shown that an amino acid sequence consisting of
6 or more His residues in a row will also act as a metal binding
site for a recombinant protein.
• A His-Tag sequence can be placed on the N-terminal of a
target protein by using vectors
Met-Gly-Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu-Val-
Pro-Arg-Gly-Ser....recombinant protein sequence

10. Key Parameters for the Operation of
IMAC
• The choice of metal ions for immobilization depends on
nature of substrate/ analyte.
• Trivalent cations ( Al3+, Ga3+, Fe3+) or tetravalent (Zr4+ ) are
prefered for capture of phosphoproteins or phosphopeptides.
• Divalent cations such as Cu2+, Ni2+, Zn2+, Co2+ are used for
purification of His- tagged proteins
• The pH is critical for initial binding and subsequent elution of
bound proteins. Typically, binding occurs at neutral or slightly
alkali pH (6.5 - 8.0), whereas elution generally occurs under
acidic environments (< 6.0).

11. Applications
 Isolation and purification of denaturing protein
 Purification of enzyme
 Purification of nucleotides
 Analysis of Protein
 Application of MCAC in other fields

12. Advantages
Two main advantages for using IMAC
• Efficiently separates His‐tagged proteins and
Phosphoproteins.
• Purification and the subsequent refolding can be done in a
single step.
• Useful in concentrating dilute samples.

13. Unique characteristics IMAC chromatography
• Often allows single‐step purification procedures
• Allowing to investigate how the different metal‐ions affect the
adsorption process without changing the matrix
• Has high protein loading capacities if compared to other
affinity chromatographic techniques
• Is useful for concentrating dilute protein solutions
• Is compatible with a number of buffers containing high ionic
strength or chaotropic components
• Generally does not affect the structure of proteins
• The use of a non‐charged IMAC column allows solutions to
become transiently sterile since all metal‐ions essential for
bacterial growth are removed by chelation

14. Disadvantages
• Presence of metal‐ions contaminates the purified
protein solution, because they may destabilize or
stabilize the protein
• Metal‐ion transfer (MIT) and the metal‐ion leakage lead
to protein loss
In order to strip off the undesired metal from the protein
and solve this problem, it is possible to use a metal‐free
chelating column packed with a strong chelating adsorbent
such as TED, or to add a chelating agent, such as EDTA, to
the collecting vials