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Affinity Precipitation of Proteins 39
were quantitatively precipitated in model experiments with bis-copper chelates.
In another study, Lilius et al. (20) described the purification of genetically
engineered galactose dehydrogenase with polyhistidine tail by metal affinity
precipitation. The histidines functioned as the affinity tail and the enzyme
could be precipitated when the bis-zinc complex with ethylene glycol-bis-(aminoethyl
ether)N,N,N´,N´-tetraacetic acid, EGTA (Zn) 2 , was added to the
protein solution. However, in general, the application of affinity precipitation
with homo-bifunctional ligands has been quite limited (21). The requirement
of a multi-binding functionality of the target protein and slow precipitation rate
restricts the use of this type of affinity precipitation process (19,22,23). The
concentration dependence and the risk of terminal aggregate formation further
complicates its use (22).
On the other hand, hetero-bifunctional format of affinity precipitation is
a more general approach, wherein affinity ligands are covalently coupled to
soluble–insoluble polymers. The ligand selectively binds the target protein
from the crude extract. The protein–polymer complex is precipitated from the
solution by a simple change of the environment property (pH, temperature, or
ionic strength). Finally, the desired protein is dissociated from the polymer,
and the latter can be recovered and reused for another cycle (18).
In metal chelating affinity precipitation, metal ligands are covalently
coupled to the reversible soluble–insoluble polymers (mainly thermoresponsive
polymers) by radical copolymerization. The copolymers carrying
metal chelating ligands are charged with metal ions and the target protein
binds the metal-loaded copolymer in solution via the interaction between the
histidine on the protein and the metal ion. The complex of the target protein
with copolymer is precipitated from the solution by increasing the temperature
in the presence of NaCl, whereas impurities remain in the supernatant
and are discarded after the separation of precipitate. The precipitated complex
is solubilized by reversing the precipitation conditions, and the target protein
is dissociated from the precipitated polymer by using imidazole or EDTA as
eluting agent. The protein is recovered from the copolymer by precipitating the
latter at elevated temperature in presence of NaCl. The metal chelating affinity
precipitation technique is presented schematically in Fig. 1. The technique
uses mainly the thermoresponsive polymers, and these polymers constitute a
major group of reversibly soluble–insoluble polymers. Among these, poly(Nisopropylacrylamide),
poly(vinyl methyl ether), and poly(N-vinylcaprolactam)
have been widely studied and used for various applications (24). Copolymers
of N-isopropylacrylamide (NIPAM) were mostly used in affinity precipitation
methods. Poly(NIPAM) has a critical temperature of precipitation at about
32°C in water and changes reversibly from hydrophilic below this temperature
to hydrophobic above it (25). This transition occurs rather abruptly at what