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42 Kumar et al.
coordination sites of the metal ion are occupied by imidazole ligands of the
polymer. The unoccupied coordination sites of the metal ion could be used for
complex formation with the protein molecule via histidine residues on its surface.
A Cu(II) charged copolymer of poly(VI-NIPAM) can also be applied for the
separation of single-stranded nucleic acids such as RNA from double-stranded
linear and plasmid DNA by affinity precipitation (31). The separation method
utilizes the interaction of metal ions to the aromatic nitrogens in exposed purines
in single-stranded nucleic acids (13–14).
Very recently, a metal affinity purification method for His-tagged proteins
based on temperature-triggered precipitation of the chemically modified elastinlike
proteins (ELPs) biopolymers have been demonstrated (16). ELPs are
biopolymers consisting of the repeating penta-peptide, VPGVG. They behave
very similar to poly(NIPAM) polymers and have been shown to undergo
reversible-phase transitions within a wide range of conditions (32,33). By
replacing the valine residue at the 4th position with a lysine in a controlled
fashion, metal-binding ligands such as imidazole can be specifically coupled
to the free amine group on the lysine residues, creating the required metal
coordination chemistry for metal affinity precipitation. ELPs with repeating
sequences of [(VPGVG) 2 (VPGKG)(VPGVG) 2 ] 21 were synthesized, and the
free amino groups on the lysine residues were modified by reacting with
imidazole-2-carboxyaldehyde to incorporate the metal-binding ligands into the
ELP biopolymers. Biopolymers charged with Ni(II) were able to interact with
a His-tag on the target proteins based on metal coordination chemistry. Purifications
of two His-tagged enzymes, -D-galactosidase and chloramphenicol
acetyltransferase, were used to demonstrate the application of metal affinity
precipitation using this new type of affinity reagent. The bound enzymes were
easily released by addition of either EDTA or imidazole. The recovered ELPs
were reused with no observable decrease in the purification performance.
Other types of metal chelating polymers for affinity precipitation of proteins
were reported recently by synthesizing highly branched copolymers of NIPAM
and 1,2-propandiol-3-methacrylate (GMA), poly(NIPAM-co-GMA) using the
technique of reversible addition fragmentation chain transfer polymerization
using a chain transfer agent that allows the incorporation of imidazole functionality
in the polymer chain ends. The LCST of the copolymers can be controlled
by the amount of hydrophobic and GMA comonomers incorporated during
copolymerization procedures. The copolymers demonstrated LCST below 18°C
and were successfully used to purify a His-tagged BRCA-1 protein fragment
by affinity precipitation (34,35).
It is important to mention here that metal chelating copolymers as discussed
above for metal chelating affinity precipitation for proteins are not yet available
commercially. Thus for carrying out affinity precipitation of proteins using