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38 Kumar et al.
protein (5), cellulose-binding domain (6), glutathione-S transferase (7), and
thioredoxin (8)) and an immobilized ligand.
The concept of using metal chelating in affinity techniques, like immobilized
metal-affinity chromatography (IMAC), was a breakthrough introduction (9).
IMAC technique has a wide application in protein purification particularly
when dealing with recombinant proteins (10,11). This offers a number of
important advantages over other “biospecific” affinity techniques for protein
purification particularly with respect to ligand stability, protein loading, and
recovery (10). The technique is generally based on the selective interaction
between metal ions like Cu(II) or Ni(II) that are immobilized on the solid
support and electron donor groups on the proteins. The amino acids histidine,
cysteine, tryptophan, and arginine have strong electron donor groups in their
side chains, and the presence of such exposed residues is an important factor
for IMA-binding properties (12). In the recombinant proteins, polyhistidine tag
(His-tag) fused to either the N- or C-terminal end of the protein has become
the selective and efficient separation tool for applying in IMAC separation.
Proteins containing a polyhistidine tag are selectively bound to the matrix,
whereas other cellular proteins are washed out. IMAC has also been utilized for
the separation of nucleic acids through the interactions of aromatic nitrogens
in exposed purines in single-stranded nucleic acids (13,14). At present, it is
one of the most popular and successful methods used in molecular biology for
the purification of recombinant proteins. The widespread application of metal
affinity concept has also recently gained usefulness by adopting the technique
in a non-chromatographic format like “metal chelating affinity precipitation”
(2,15–17). Such separation strategy makes metal affinity methods more simple
and cost-effective when the intended applications are for large-scale processes.
This chapter discusses affinity precipitation method using metal chelating
polymers for selective separation of proteins. Affinity precipitation is a
relatively new technique, which allows protein separation from crude
homogenates with rather high yields compared to conventional chromatography
(18). By combining the versatile properties of metal affinity with affinity precipitation,
the technique presents enormous potential as a simple and selective
separation strategy. Affinity precipitation methods have two main approaches
that have been described in the literature (18), namely, precipitation with homoor
hetero-bifunctional ligands. Previously, there have been a few attempts to
utilize the metal affinity concept in affinity precipitation methods in homobifunctional
format. The addition of a bis-ligand at an optimum concentration
creates a cross-linked network with the target protein provided the latter has
two or more metal-binding sites. The cross-linked protein–bis–ligand network
precipitates from the solution eventually. The first such application was reported
by Van Dam et al. (19) when human hemoglobin and sperm whale hemoglobin