1. Metal Chelate Affinity Chromatography Wenbo Dong Yagmur Yagdiran

2. 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.

3. 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 hydrophobicityExpanded bed adsorption (EBA) follows the principles of AC, IEX or HIC

4. 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.

5. 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.

6. 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

7. 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

8. 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).

9. 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

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

11. 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

12. 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

13. 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