Metal ChelateAffinityChromatography WenboDong YagmurYagdiran
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.
Affinity chromatography separates proteins on the basis of a reversible interaction betweena protein (or group of proteins) and a specific ligand coupled to a chromatography matrix.
Metal-ChelateAffinityChromatography • Metal-Chelate Affinity Chromatography (MCAC), also knownas Immobilized Metal Affinity Chromatography (IMAC), wasfirst successfully demonstrated in 1975 byPorath andcollaborators for human serum proteins.
MCAC commonlyutilizeszinc (Zn2+), nickel (Ni2+) orcopper (Cu2+) to form stablecomplexeswith histidine, tryptophan and cysteine residues within proteins. • Once bound, the proteins can be eluted via pH or imidazolegradients
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 MetGlySerSerHisHisHisHisHisHisSerSerGlyLeuValProArgGlySer....recombinant protein sequence
Key Parameters for the Operation of MCAC • Chelatingagents, suchas ethylenediaminetetraceticacid (EDTA) andethyleneglycolbis(β-aminoethylether) N, N, N’, N’,-tetraaceticacid (EGTA), must beexcluded from all solutions because they willstripthe metal ions from the matrix. • The pH is critical for initial binding and subsequentelution of bound proteins. Typically, binding occurs at neutralor slightly alkali pH (6.5 - 8.0), whereas elution generallyoccurs 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 = QmaxKc/ 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 contaminatsthe 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