Transcript Metal chelate chrom

Metal Chelate Affinity
Chromatography
Wenbo Dong
Yagmur Yagdiran
• Protein purification is a series of processes
intended to isolate a single type of protein
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 N-terminal of a
target protein by using vectors
MetGlySerSerHisHisHisHisHisHisSerSerGlyLeuVa
lProArgGlySer....recombinant protein sequence
Key Parameters for the Operation of
MCAC
• Chelating agents, such as ethylenediaminetetracetic
acid (EDTA) and ethylene glycolbis(β-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, it’s 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 His‐tagged proteins in the presence of
denaturing concentrations of urea and guanidine‐HCl
• purification and the subsequent refolding can be done in a
single step
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
Disadvantages
• the presence of metal‐ions contaminats the purified protein
solution, because they may whether 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