advances-in-protein-chemistry

As mentioned earlier, the interaction used in IMAC depends mainly on the formation of coordinate bonds between electron deficient
metal ions and the electron donor groups on the protein surface. Side chains of certain amino acids are differentially suitable for binding.
Histidine exhibits the strongest interaction, due to the presence of imidazole ring that readily forms coordination bonds with metal ions.
To a lesser extent, sulfhydryl group of Cysteine, and aromatic side groups of Tryptophan, Tyrosine and Phenylalanine can also contribute
to binding. However, the actual retention of protein is primarily based on the presence of histidine residues.
Since most of the proteins contain these amino acids in varying amounts, all proteins should theoretically be capable of binding to
immobilized metal. However, the actual strength of binding would depend on the number of these residues as well as their presence on
the surface of protein.
Target protein can be eluted from an IMAC resin decreasing the pH to 4-5 which leads to the protonation of histidine’s imidazole
group and hence the dissociation of the protein. For proteins that are sensitive to acidic pH, competitive elution with imidazole at nearly
neutral pH is recommended.
Although, IMAC cannot be considered as highly specific when compared to other techniques, it is atleast moderately specific.
However, it offers advantages such as stability of ligand, high capacity, mild elution conditions, ease of regeneration and low cost makes
it useful.
IMAC holds an advantage over biospecific affinity techniques due to its structure-independent interaction that makes it effective even
under denaturing conditions. This is often desirable when proteins are expressed in E. coli in the form of inclusion bodies.
On the other hand, IMAC may not be the technique of choice for the production of therapeutic proteins, due to problems with
reproducibility, and contamination by host cell proteins, toxins, viruses etc. Moreover, metal ion leaching from the sorbent can cause
damage to the target proteins by metal-catalyzed reactions.
Immuno-Affinity chromatography and immunoprecipitation: Immuno-affinity chromatography, also known as immuneadsorption
chromatography, is a specialized form of affinity chromatography. It is based on the highly specific antigen-antibody
interactions, and utilizes an antibody (or antibody fragment) immobilized onto a solid support matrix as a ligand in a manner that
it retains its binding capacity towards the antigen. The crude sample is passed through the column where the binding of protein with
antibody occurs. The unbound material is then washed clear before the elution of the retained protein. The elution is performed by
alterations of the buffer (mobile phase) conditions to weaken the antibody-antigen interaction. The technique may well be used for the
separation of antibodies using immobilized antigen.
Immunoprecipitation is also based on antigen-antibody interactions and therefore it is, in principle, similar to Immuno-affinity
chromatography. The affinity of Protein-A or Protein-G for the antibodies is usually exploited. In the pre-immobilized antibody approach
(Figure 3 A), the target protein (antigen) is allowed to bind to its antibody that is pre-bound to Protein-A (or Protein-G) attached to
a solid support (beads). Alternatively, free antibody approach may be used that where the attachment to Protein-A (or Protein-G)
beads is carried out after the formation of antigen-antibody complex (Figure 3 B). In either case, the beads are then separated from the
mixture by centrifugation and the antigen is retrieved. The technique may also be used for pulling down and isolating the whole protein
complex if the target protein naturally exists in association with other known or unknown protein partners, and is often termed as Co-
Immunoprecipitation in this case. However, care should be exercised in interpretation of results since sometimes some non-specifically
associated proteins may also get pulled along with the target protein.
Figure 3: Steps in Immunoprecipitation. A. Pre-immobilized antibody approach B. Free antibody approach.
Ion exchange chromatography: Ion exchange chromatography (IEC) involves the reversible interactions between a charged
protein and the chromatography medium with an opposite charge. The ion exchangers that were put in use initially were comprised of
hydrophobic polymer matrices, heavily substituted with ionic groups. The low permeability of these matrices limited their use with larger
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