Immobilized Metal affinity-chromatography
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Transcript of Immobilized Metal affinity-chromatography
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Immobilized-Metal Affinity
Chromatography
Mahendra G. Jadhao16 Nov.2015
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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 theprinciples of AC, IEX or HIC
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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.
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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.
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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.
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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
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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
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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.
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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
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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).
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Applications
Isolation and purification of denaturing protein
Purification of enzyme
Purification of nucleotides
Analysis of Protein
Application of MCAC in other fields
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AdvantagesTwo 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.
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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
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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
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Thank you!