specifically with tagged proteins carrying polyhistidine sequences [26]. As another
example, lectins are ligands able to recognize specific glycan structures and, thus, are
used for the separation of groups of glycoproteins [22]. On the same line, aptamer
ligands can be designed for the recognition of similar structures and thus used for the
separation of groups of proteins sharing similar aptatopes.
Already in 2011, Kokpinar et al. reported separation results of His-tagged
recombinant proteins using anti-His-tag aptamers [125]. They were used conventionally after the immobilization of the aptamer, previously activated and then
attached on amino-derived magnetic beads. Starting from crude extracts of E. coli
culture, the tagged protein adsorption was loaded under physiological conditions and
the recombinant protein desorbed using histidine at a concentration of 1 M in the
same buffer. Three examples of protein purifications up to almost homogeneity
were described: Pseudomonas fluorescens esterase I, Bacillus subtilis pnitrobenzylesterase, and Bacillus stearothermophilus esterase. The comparison
with classical IMAC affinity chromatography demonstrated that the purity reached
with the selected aptamer ligand was similar or better, while avoiding the involvement of questionable metal ions. In another development strategy, Bartnicki et al.
[153] showed that anti-His DNA aptamers could be used in various cases. Selected
aptamers had an affinity for the tagged proteins dependent on the presence of sodium
ions. This property allowed circumventing the necessity of histidine as eluting agent.
The aptamer, biotinylated in 5
0 , was immobilized on a streptavidin agarose support.
The adsorption of the crude protein extract was performed in the presence of
300 mM sodium chloride. After washing, captured proteins were released by the
same buffer solution but in the absence of sodium chloride. Three His-tagged
recombinant proteins expressed in E. coli were purified: proliferating cell nuclear
antigen, glutathione S-transferase, and green fluorescent protein. The authors
reported a better purity compared to Ni-NTA chromatography over repeated cycles.
Another interesting example is given by the purification of L-selectin. This is a
transmembrane receptor recognizing a tetrasaccharide ligand, expressed on the
surface of white blood cells [122]. After selection, the biotinylated specific aptamer
was coupled to streptavidin agarose beads. The recombinant protein was expressed
as His-tag, and its purification was comparatively performed using aptamer affinity
chromatography and NTA affinity chromatography. The protein adsorption phase
was under physiological conditions (PBS containing 0.9 mM CaCl 2 and 0.5 mM
MgCl 2 ). After washing, the elution of the target protein from the aptamer column
was obtained by adding 100 mM EDTA to the PBS buffer. The elution from NTA
column was performed with current procedures, involving the use of imidazole at
a concentration of 250 mM. Comparative results demonstrated the superiority of
aptamer affinity chromatography in terms of purity, yield, and binding capacity of
the affinity sorbent. These results are not surprising since the aptamer is considered
specific for the target protein, whereas the Ni-NTA displays its affinity not only for
the His-tagged proteins but also to other proteins when they expose histidine groups
on their surface.
Group-specific DNA aptamers for the separation of polypeptides sharing the
same phosphorylation site were also described [154]. In the same logic, groupAptamer-Based Affinity Chromatography for Protein Extraction and Purification
123
example, lectins are ligands able to recognize specific glycan structures and, thus, are
used for the separation of groups of glycoproteins [22]. On the same line, aptamer
ligands can be designed for the recognition of similar structures and thus used for the
separation of groups of proteins sharing similar aptatopes.
Already in 2011, Kokpinar et al. reported separation results of His-tagged
recombinant proteins using anti-His-tag aptamers [125]. They were used conventionally after the immobilization of the aptamer, previously activated and then
attached on amino-derived magnetic beads. Starting from crude extracts of E. coli
culture, the tagged protein adsorption was loaded under physiological conditions and
the recombinant protein desorbed using histidine at a concentration of 1 M in the
same buffer. Three examples of protein purifications up to almost homogeneity
were described: Pseudomonas fluorescens esterase I, Bacillus subtilis pnitrobenzylesterase, and Bacillus stearothermophilus esterase. The comparison
with classical IMAC affinity chromatography demonstrated that the purity reached
with the selected aptamer ligand was similar or better, while avoiding the involvement of questionable metal ions. In another development strategy, Bartnicki et al.
[153] showed that anti-His DNA aptamers could be used in various cases. Selected
aptamers had an affinity for the tagged proteins dependent on the presence of sodium
ions. This property allowed circumventing the necessity of histidine as eluting agent.
The aptamer, biotinylated in 5
0 , was immobilized on a streptavidin agarose support.
The adsorption of the crude protein extract was performed in the presence of
300 mM sodium chloride. After washing, captured proteins were released by the
same buffer solution but in the absence of sodium chloride. Three His-tagged
recombinant proteins expressed in E. coli were purified: proliferating cell nuclear
antigen, glutathione S-transferase, and green fluorescent protein. The authors
reported a better purity compared to Ni-NTA chromatography over repeated cycles.
Another interesting example is given by the purification of L-selectin. This is a
transmembrane receptor recognizing a tetrasaccharide ligand, expressed on the
surface of white blood cells [122]. After selection, the biotinylated specific aptamer
was coupled to streptavidin agarose beads. The recombinant protein was expressed
as His-tag, and its purification was comparatively performed using aptamer affinity
chromatography and NTA affinity chromatography. The protein adsorption phase
was under physiological conditions (PBS containing 0.9 mM CaCl 2 and 0.5 mM
MgCl 2 ). After washing, the elution of the target protein from the aptamer column
was obtained by adding 100 mM EDTA to the PBS buffer. The elution from NTA
column was performed with current procedures, involving the use of imidazole at
a concentration of 250 mM. Comparative results demonstrated the superiority of
aptamer affinity chromatography in terms of purity, yield, and binding capacity of
the affinity sorbent. These results are not surprising since the aptamer is considered
specific for the target protein, whereas the Ni-NTA displays its affinity not only for
the His-tagged proteins but also to other proteins when they expose histidine groups
on their surface.
Group-specific DNA aptamers for the separation of polypeptides sharing the
same phosphorylation site were also described [154]. In the same logic, groupAptamer-Based Affinity Chromatography for Protein Extraction and Purification
123