Metal Chelate

Affinity
Chromatography
Wenbo Dong
Yagmur Yagdiran

 Protein purificationis a series of

processes intended to isolate a single
type ofprotein from a complex
mixture.
Protein purification is vital for the
characterization of the function,
structure and interactions of the
protein of interest.

Proteins are purified using

chromatographic purification
techniques which separate according to
differences in 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

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.

Metal-Chelate 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.

 MCAC commonly utilizes zinc

(Zn2+), nickel (Ni2+) or copper
(Cu2+) to form stable complexes
with histidine, tryptophan and
cysteine residues within proteins.
Once bound, the proteins can be
eluted via pH or imidazole
gradients

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 Nterminal of a target protein by using vectors

MetGlySerSerHisHisHisHisHisHisSer
SerGlyLeuValProArgGlySer....recomb
inant protein sequence

Key Parameters for the

Operation of MCAC
Chelating agents, such as
ethylenediaminetetracetic acid (EDTA) and
ethylene glycolbis(-aminoethyl ether) N, N,
N, N,-tetraacetic acid (EGTA), must be
excluded from all solutions because they will
strip the metal ions from the matrix.
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).

Theory Model
Exact model has not been established.
Langmuir Model
P + n Cu

==== P Cu + (n-1) Cu

K1 = [P Cu] / [P] [Cu]

When the binding reach the balance: Q = Qmax Kc/
1+ Kc
In reality, its quit rare for the situation that one
protein has only one binding site, therefore, based on
the it, models of multiple sites have been established
and accepted. Like Freunlium model, Temkin model,
Langmuir-Freunlich model and Double-Langmuir
model

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

Advantages
Two main advantages for using IMAC
Efficiently separating Histagged proteins in the
presence of denaturing concentrations of urea
and guanidineHCl
purification and the subsequent refolding can be
done in a single step

Unique characteristics IMAC

chromatography

Often allows singlestep purification procedures

Allowing to investigate how the different metalions
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 noncharged IMAC column allows
solutions to become transiently sterile since all
metalions essential for bacterial growth are
removed by chelation

Disadvantag
es
the presence of metalions contaminats the purified
protein solution, because they may whether
destabilize or stabilize the protein

metalion transfer (MIT) and the metalion 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 metalfree
chelating column packed with a strong chelating
adsorbent such as TED, or to add a chelating agent,
such as EDTA, to the collecting vials

ank you for your attentio